USPA1 and USPA2 antigens of Moraxella catarrhalis

ABSTRACT

The present invention discloses the existence of two novel proteins UspA1 and UspA2, and their respective genes uspA1 and uspA2. Each protein encompasses a region that is conserved between the two proteins and comprises an epitope that is recognized by the MAb 17C7. One or more than one of these species may aggregate to form the very high molecular weight form (i.e. greater than 200 kDa) of the UspA antigen. Compositions and both diagnostic and therapeutic methods for the treatment and study of  M. catarrhalis  are disclosed.

This is a continuation of co-pending application Ser. No. PCT/US97/23930filed Dec. 19, 1997, which claims priority to U.S. provisional patentapplication Ser. No. 60/033,598, filed Dec. 20, 1996.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under NIH Grant NumberAI23366. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates generally to the fields of microbiology,and clinical bacteriology. More particularly, it concerns sequences ofthe uspA1 and uspA2 genes which encode the proteins UspA1 and UspA2,respectively, both of which encode an epitope reactive with monoclonalantibody (MAb) 17C7 and provide useful epitopes for immunodiagnosis andimmunoprophylaxis.

II. Description of Related Art

It was previously thought that Moraxella catarrhalis, previously knownas Branhamella catarrhalis or Neisseria catarrhalis, was a harmlesssaprophyte of the upper respiratory tract (Catlin, 1990; Berk, 1990).However, during the previous decade, it has been determined that thisorganism is an important human pathogen. Indeed, it has been establishedthat this Gram-negative diplococcus is the cause of a number of humaninfections (Murphy, 1989). M. catarrhalis is now known to be the thirdmost common cause of both acute and chronic otitis media (Catlin, 1990;Faden et al., 1990; 1991; Marchant, 1990), the most common disease forwhich infants and children receive health care according to the 1989Consensus Report. This organism also causes acute maxillary sinusitis,generalized infections of the lower respiratory tract (Murphy and Loeb,1989) and is an important cause of bronchopulmonary infections inpatients with underlying chronic lung disease and, less frequently, ofsystemic infections in immunocompromised patients (Melendez and Johnson,1990; Sarubbi et al., 1990; Schonheyder and Ejlertsen, 1989; Wright andWallace, 1989).

The 1989 Consensus Report further concluded that prevention of otitismedia is an important health care goal due to both its occurrence ininfants and children, as well as certain populations of all age groups.In fact, the total financial burden of otitis media has been estimatedto be at least $2.5 billion annually. Vaccines were identified as themost desired approach to prevent this disease for a number of reasons.For example, it was estimated that if vaccines could reduce theincidence of otitis media by 30%, then the annual health care savingswould be at least $400 million. However, while some progress has beenmade in the development of vaccines for 2 of the 3 common otitis mediapathogens, Streptococcus pneumoniae and Haemophilus influenzae, there isno indication that similar progress has been made with respect to M.catarrhalis. This is particularly troublesome in that M. catarrhalis nowaccounts for approximately 17-20% of all otitis media infection (Murphy,1989). In addition, M. catarrhalis is also a significant cause ofsinusitis (van Cauwenberge et al., 1993) and persistent cough (Gottfarband Brauner, 1994) in children. In the elderly, it infects patients withpredisposing conditions such as chronic obstructive pulmonary disease(COPD) and other chronic cardiopulmonary conditions (Boyle et al., 1991;Davies and Maesen, 1988; Hager et al., 1987).

Despite its recognized virulence potential, little is known about themechanisms employed by M. catarrhalis in the production of disease orabout host factors governing immunity to this pathogen. An antibodyresponse to M. catarrhalis otitis media has been documented by means ofan ELISA system using whole M. catarrhalis cells as antigen and acuteand convalescent sera or middle ear fluid as the source of antibody(Leinonen et al., 1981). The development of serum bactericidal antibodyduring M. catarrhalis infection in adults was shown to be dependent onthe classical complement pathway (Chapman et al., 1985). And morerecently, it was reported that young children with M. catarrhalis otitismedia develop an antibody response in the middle ear but fail to developa systemic antibody response in a uniform manner (Faden et al., 1992).

Previous attempts have been made to identify and characterize M.catarrhalis antigens that would serve as potentially important targetsof the human immune response to infection (Murphy, 1989; Goldblatt etal., 1990; Murphy et al., 1990). Generally speaking, the surface of M.catarrhalis is composed of outer membrane proteins (OMPs),lipooligosaccharide (LOS) and fimbriae. M. catarrhalis appears to besomewhat distinct from other Gram-negative bacteria in that attempts toisolate the outer membrane of this organism using detergentfractionation of cell envelopes has generally proven to be unsuccessfulin that the procedures did not yield consistent results (Murphy, 1989;Murphy and Loeb, 1989). Moreover, preparations were found to becontaminated with cytoplasmic membranes, suggesting an unusualcharacteristic of the M. catarrhalis cell envelope.

Passive immunization with polyclonal antisera raised against outermembrane vesicles of the M. catarrhalis strain 035E was also found toprotect against pulmonary challenge by the heterologous M. catarrhalisstrain TTA24. In addition, active immunization with M. catarrhalis outermembrane vesicles resulted in enhanced clearance of this organism fromthe lungs after challenge. The positive effect of immunization inpulmonary clearance indicates that antibodies play a major role inimmunoprotection from this pathogen. In addition, the protectionobserved against pulmonary challenge with a heterologous M. catarrhalisstrain demonstrates that one or more conserved surface antigens aretargets for antibodies which function to enhance clearance of M.catarrhalis from the lungs.

Outer membrane proteins (OMPs) constitute major antigenic determinantsof this unencapsulated organism (Bartos and Murphy, 1988) and differentstrains share remarkably similar OMP profiles (Bartos and Murphy, 1988;Murphy and Bartos, 1989). At least three different surface-exposed outermembrane antigens have been shown to be well-conserved among M.catarrhalis strains; these include the 81 kDa CopB OMP (Helminen et al.,1993b), the heat-modifiable CD OMP (Murphy et al., 1993) and thehigh-molecular weight UspA antigen (Helminen et al., 1994). Of thesethree antigens, both the CopB protein and UspA antigen have been shownto bind antibodies which exert biological activity against M.catarrhalis in an animal model (Helminenet al., 1994; Murphy et al.,1993).

The MAb, designated 17C7, was described as binding to UspA, a very highmolecular weight protein that migrated with an apparent molecular weight(in SDS-PAGE) of at least 250 kDa (Helminen et al., 1994; Klingman andMurphy, 1994). MAb 17C7 enhanced pulmonary clearance of M. catarrhalisfrom the lungs of mice when used in passive immunization studies and, incolony blot radioimmunoassay analysis, bound to every isolate of M.catarrhalis examined. This same MAb also reacted, although lessintensely, with another antigen band of approximately 100 kDa, asdescribed in U.S. Pat. No. 5,552,146 (incorporated herein by reference).A recombinant bacteriophage that contained a fragment of M. catarrhalischromosomal DNA that expressed a protein product that bound MAb 17C7 wasalso identified and migrated at a rate similar or indistinguishable fromthat of the native UspA antigen from M. catarrhalis (Helminen et al.,1994).

With the rising importance of this pathogen in respiratory tractinfections, identification of the surface components of this bacteriuminvolved in virulence expression and immunity is becoming moreimportant. To date, there are no vaccines available, against any otherOMP. LOS or fimbriac, that induce protective antibodies against M.catarrhalis. Thus, it is clear that there remains a need to identify andcharacterize useful antigens and which can be employed in, thepreparation of immunoprophylatic reagents. Additionally, once such anantigen or antigens is identified, there is a need for providing methodsand compositions which will allow the preparation of vaccines and inquantities that will allow their use on a wide scale basis inprophylactic protocols.

SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to provide newUspA1 and UspA2 proteins and genes coding therefor. It also is an objectof the present invention to provide methods of using these new proteins,for example, in the preparation of agents for the treatment andinhibition of M. catarrhalis infection. It also is contemplated thatthrough the use of other technologies such as antibody treatment andimmunoprophylaxis that one can inhibit or even. prevent M. catarrhalisinfections.

In satisfying these goals, there are provided epitopic core sequences ofUspA1 and UspA2 which can serve as the basis for the preparation oftherapeutic or prophylactic compositions or vaccines which comprisepeptides of 7, 10, 20, 30, 40, 50 or even 60 amino acids in length thatelicit an antigenic reaction and a pharmaceutically acceptable buffer ordiluent. These peptides may be coupled to a carrier, adjuvant, anotherpeptide or other molecule such that an effective antigenic response toM. catarrhalis is retained or even enhanced. Alternatively, thesepeptides may act as carriers themselves when coupled to another peptideor other molecule that elicits an antigenic response to M. catarrhalisor another pathogen. For example, UspA2 can serve as a carrier for anoligosaccharide.

In one embodiment, the epitopic core sequences of UspA1 and UspA2comprise one or more more isolated peptides of 7, 10, 20, 30, 40, 50 oreven 60 amino acids in length having the amino acid sequence AQQQDQH(SEQ ID NO:17).

In another embodiment, there are provided nucleic acids, uspA1 anduspA2, which encode the UspA1 and the UspA2 antigens, respectively, aswell as the amino acid sequences of the UspA1 and UspA2 antigens of theM. catarrhalis isolates O35E, TTA24, TTA37, and O46E. It is envisionedthat nucleic acid segments and fragments of the genes uspA1 and uspA2and the UspA1 and UspA2 antigens will be of value in the preparation anduse of therapeutic or prophylactic compositions or vaccines fortreating, inhibiting or even preventing M. catarrhalis infections.

In another embodiment, there is provided a method for inducing an immuneresponse in a mammal comprising the step of providing to the mammal anantigenic composition that comprises an isolated peptide of about 20 toabout 60 amino acids that contains the identified epitopic core sequenceand a pharmaceutically acceptable buffer or diluent.

In another embodiment, there is provided a method for diagnosing M.catarrhalis infection which comprises the step of determining thepresence, in a sample, of an M. catarrhalis amino acid sequencecorresponding to residues of the epitopic core sequences of either theUspA1 or UspA2 antigen. This method may comprise PCR m detection of thenucleotide sequences or alternatively an immunologic reactivity of anantibody to either a UspA1 or UspA2 antigen.

In a further embodiment, there is provided a method for treating anindividual having an M. catarrhalis infection which comprises providingto the individual an isolated peptide of about 20 to about 60 aminoacids that comprises at least about 10 consecutive residues of the aminoacid sequence identified as an epitopic core sequence of UspA1 or UspA2.

In a still further embodiment, there is provided a method for preventingor limiting an M. catarrhalis infection that comprises providing to asubject an antibody that reacts immunologically with the identifiedepitopic core region of either UspA1 or UspA2 of M. catarrhalis.

In another embodiment, there is provided a method for screening apeptide for reactivity with an antibody that binds immunologically toUspA1, UspA2 or both which comprises the steps of providing the peptideand contacting the peptide with the antibody and then determining thebinding of the antibody to the peptide. This method may comprise animmunoassay such as a western blot, an ELISA, an RIA or animmunoaffinity separation.

In a still further embodiment, there is provided a method for screeninga UspA1 or UspA2 peptide for its ability to induce a protective immuneresponse against M. catarrhalis by providing the peptide, administeringit in a suitable form to an experimental animal, challenging the animalwith M. catarrhalis and then assaying for an M. catarrhalis infection inthe animal. It is envisioned that the animal used will be a mouse thatis challenged by a pulmonary exposure to M. catarrhalis and that theassaying comprises assessing the degree of pulmonary clearance by themouse.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples, while indicating preferred embodiments of the invention, aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIG. 1. Southern blot analysis of PvuII-digested chromosomal DNA fromstrains of M. catarrhalis using a probe from the uspA1 gene. Bacterialstrain designations are at the top; kilobase (kb) position markers areon the left.

FIG. 2A. Proteins present in whole cell lysates of the wild-type strainO35E and the isogenic uspA1 mutant strain. These proteins were resolvedby SDS-PAGE and stained with Coomassie blue. The left lane (WT) containsthe wild-type strain and right lane (MUT) contains the mutant. Thearrows indicate the protein, approximately 120 kDa in size, that ispresent in the wild-type and missing in the mutant. Kilodalton positionmarkers are on the left.

FIG. 2B. Western blot analysis of whole cell lysates of the wild-typestrain O35E and the isogenic uspA1 mutant strain. These proteins wereresolved by SDS-PAGE and probed with MAb 17C7 in western blot analysis.The left lane (WT) contains the wild-type strain and the right lane(MUT) contains the mutant. Kilodalton position markers are on the left.It can been seen that both strains possess the very high molecularweight band reactive with MAb 17C7 whereas only the wild-type strainalso has a band of approximately 120 kDa that binds this MAb.

FIG. 2C. Western blot analysis of whole cell lysate (WCL) andEDTA-extracted outer membrane vesicles (OMV) from the wild-type strainO35E (WT) and the isogenic uspA1 mutant (MUT) using MAb 17C7. Sampleswere either heated at 37° C. for 15 minutes (H) or at 100° C. forminutes (B) prior to SDS-PAGE. Molecular weight position markers (inkilodaltons) are indicated on the left. The open arrow indicates theposition of the very high molecular weight form of the MAb 17C7-reactiveantigen; the closed arrow indicates the position of the approximately120 kDa protein; the open circle indicates the position of theapproximately 70-80 kDa protein.

FIG. 3. Southern blot analysis of chromosomal DNA from the wild-type M.catarrhalis strain O35E and the isogenic uspA1 mutant. Chromosomal DNAwas digested with PvuII and probed with a 0.6 kb BglII-PvuII fragmentfrom the uspA1 gene. The wild-type strain is listed as O35E at the topof this figure and the mutant strain is listed as O35E-uspA1⁻. Kilobaseposition markers are present on the left side.

FIG. 4. Western blot reactivity of proteins in M. catarrhalis strainO35E outer membrane vesicles (labeled O35E OMV) and the MF-4-1 GSTfusion protein (labeled GST fusion protein) with MAb 17C7.

FIG. 5. PCR™ products obtained by the use of the T3 and P10 primers(middle lane—0.9 kb product) and the T7 and P9 primers (right lane—1.7kb product) when used in a PCR™ amplification with chromosomal DNA fromthe uspA1 mutant. A kb ladder is present in the first lane; several kbposition markers are listed on the left side of this figure.

FIGS. 6A-6C. SDS-PAGE and westerns of purified proteins. FIG. 6A.Coomassie blue stained gel of purified UspA2 (lane 2). FIG. 6B.Coomassie blue stained gel of purified UspA1 prepared without heating ofsample (lane 4), heated for 3 min at 100° C. (lane 5), heated for 5 minat 100° C. (lane 6), and heated for 10 min at 100° C. (lane 7). FIG. 6C.Western of the purified UspA2 (lane 9) and purified UspA1 (lane 10)probed with the 17C7 MAb. Both proteins were heated 10 min. Themolecular size markers in lanes 1, 3, and 8 are as indicated inkilodaltons.

FIG. 7. Interaction of purified UspA1 and UspA2 with HEp-2 cells asdetermined by ELISA. HEp-2 cell monolayers cultured in 96-well platewere incubated with serially diluted UspA1 or UspA2. O35E bacterialstrain was used as the positive control. The bacteria were dilutedanalogous to the proteins beginning with a suspension with an A₅₅₀ of1.0. The bound proteins or attached bacteria were detected with a 1:1mixed antisera to UspA1 and UspA2 as described in the methods.

FIG. 8. Interaction with fibronectin and vitronectin determined by dotblot. The bound vitronectin was detected with rabbit polyclonalantibodies, the protein bound to the fibronectin was detected withpooled sera made against the UspA1 and UspA2.

FIG. 9. The levels of antibodies to the protein UspA1, UspA2 and M.catarrhalis O35E strain in normal human sera. Data are the log₁₀transformed end-point titers of the IgG (FIGS. 9A-9C) and IgA (FIGS.9D-9F) antibodies determined by ELISA. The individual titers wereplotted according to age group and the geometric mean titer for each agegroup linked by a solid line. Sera for the 2-18 month old children wereconsecutive samples from a group of ten children.

FIG. 10. Subclass distribution of IgG antibodies to UspA1 and UspA2 innormal human sera. FIG. 10A shows titers toward UspA1 and FIG. 10B showstiters to UspA2.

FIG. 11. Relationship of serum IgG titers to UspA1 (FIG. 11A) and UspA2(FIG. 11B) with the bactericidal liter against the O35E straindetermined by logistic regression (p<0.05). The solid line indicates thelinear relationship between the IgG titer and bactericidal titer. Brokenlines represent the 95% confidence intervals of the linear fit.

FIG. 12. Schematic drawing showing the relative positions ofdecapeptides 10-24 within the region of UspA1 and UspA2 which binds toMAb 17C7.

FIG. 13. Western dot blot analysis demonstrating reactivity ofdecapeptides 10-24 with MAb 17C7.

FIG. 14. Partial restriction enzyme map of the uspA1 (FIG. 14A) anduspA2 (FIG. 14B) genes from M. catarrhalis strain O35E and the mutatedversions of these genes. The shaded boxes indicate the open readingframe of each gene. Relevant restriction sites are indicated. PCR™primer sites (P1-P6) are indicated by arrows. The DNA fragmentscontaining the partial uspA1 and uspA2 open reading frames that werederived from M. catarrhalis straina O35E chromosomal DNA by PCR™ andcloned into pBluescriptII SK+ are indicated by black bars. Dotted linesconnect corresponding restriction sites on these DNA inserts and thechromosome. Open bars indicate the location of the kanamycin orchloramphenicol cassettes, respectively. The DNA probes specific foruspA1 or uspA2 are indicated by the appropriate cross-hatched bars andwere amplified by PCR™ from M. catarrhalis strain O35E chromosomal DNAby the use of the oligonucleotide primer pairs

P3 (5′-GACGCTCAACAGCACTAATACG-3′) (SEQ ID NO:20)/P4(5′-CCAAGCTGATATCACTACC-3′) (SEQ ID NO:21) and

P5 (5′-TCAATGCCMTTGATGGTC-3′) (SEQ ID NO:22)/P6(5′-TGTATGCCGCTACTCGCAGCT-3′) (SEQ ID NO:23), respectively.

FIG. 15. Detection of the UspA1 and UspA2 proteins in wild-type andmutant strains of M. catarrhalis O35E. Proteins present inEDTA-extracted outer membrane vesicles from the wild-type strain (lane1), the uspA1 mutant strain O35E.1 (lane 2), the uspA2 mutant strainO35E.2 (lane 3), and the isogenicuspA1 uspA2 double mutant strainO35E.12 (lane 4) were resolved by SDS-PAGE, and either stained withCoomassie blue (FIG. 15A) or transferred to nitrocellulose and probedwith MAb 17C7 followed by radioiodinated goat anti-mouse immunoglobulinin western blot analysis. In FIG. 15A, the closed arrow indicates thevery high molecular weight form of the UspA antigen which is comprisedof both UspA1 and UspA2. In FIG. 15B, the bracket on the left indicatesthe very high molecular weight forms of the UspA1 and UspA2 proteinsthat bind MAb 17C7. The open arrow indicates the 120 kDa, putativemonomeric form of UspA1. The closed arrow indicates the 85 kDa, putativemonomeric form of UspA2. Molecular weight position markers (inkilodaltons) are present on the left.

FIG. 16. Comparison of the rate and extent of growth Qf the wild-typeand mutant strains of M. catarrhalis. The wild-type strain O35E (closedsquares), the uspA1 mutant O35E.1 (open squares), the uspA2 mutantO35E.2 (closed circles), and the uspA1 uspA2 double mutant O35E.12 (opencircles) of M. catarrhalis O35E from overnight broth cultures werediluted to a density of 35 Klett units in BHI broth and subsequentlyallowed to grow at 37° with shaking. Growth was followed by means ofturbidity measurements.

FIG. 17. Susceptibility of wild-type and mutant strains of M.catarrhalis to killing by normal human serum. Cells of the wild-typeparent strain O35E (diamonds), uspA1 mutant O35E.1 (triangles), uspA2mutant O35E.2 (circles), and uspA1 uspA2 double mutant O35E.12 (squares)from logarithmic-phase BHI broth cultures were incubated in the presenceof 10% (v/v) normal human serum (closed symbols) or heat-inactivatednormal human serum (open symbols). Data are presented as the percentageof the original inoculum remaining at each time point.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

The present invention relates to the identification of epitopes usefulfor developing potential vaccines against M. catarrhalis. Early work wasdirected at determining the molecular nature of the UspA antigen andcharacterize the epitope which is recognized by the MAb 17C7.Preliminary work indicated that MAb 17C7 recognizes a single antigenicepitope and it was believed that this epitope was encoded by a singlegene. However, isolation of the protein which contained the epitopeyielded unexpected results. MAb 17C7 recognized a single epitope, butthe characteristics of the protein associated with the epitope suggestedthe existence of not one but two separate proteins. Further carefulanalyses led to a surprising discovery. A single epitope of the UspAantigen is recognized by the MAb 17C7, but this epitope is present intwo different proteins, UspA1 and UspA2, which are encoded by twodifferent genes uspA1 and uspA2, respectively, and only have 43%identity to each other. The present invention provides the nucleotidesequences of the genes uspA1 and uspA2, their respective proteinproducts, UspA1 and UspA2, and the shared epitope recognized by MAb17C7.

In addition, the present invention provides insights into the antigenicstructure of the UspA protein based on the analysis of the sequences ofthe UspA1 and UspA proteins which comprise the protein. Characterizationof the epitopic region of the molecule that is targeted by the MAb 17C7permits the development of agents that will be useful in protectingagainst M. catarrhalis infections, e.g., in the preparation ofprophylactic reagents. Particular embodiments relate to the amino acidand nucleic acids corresponding to the UspA1 and UspA2 proteins,peptides and antigenic compositions derived therefrom, and methods forthe diagnosis and treatment of M. catarrhalis disease.

As stated previously, M. catarrhalis infections present a serious healthchallenge, especially to the young. Thus, there is a clear need todevelop compositions and methods that will aid in the treatment anddiagnosis of this disease. The present invention, by virtue of newinformation regarding the structure of the UspA antigen of M.catarrhalis, and discovery of the two new and distinct proteins UspA1and UspA2 provides such improved compositions and methods. UspA1 andUspA2 represent important antigenic determinants, as the MAb 17C7 hasbeen shown to protect experimental animals, as measured in a pulmonaryclearance model, when provided in passive immunizations.

In a first embodiment, the present invention provides for theidentification of the proteins UspA1 and UspA2 from M. catarrhalisstrain O35E. The UspA1 protein comprises about 831 amino acid residuesand has a predicted mass of about 88,271 daltons (SEQ ID NO:1). TheUspA2 protein comprises about 576 residues and has a predicted mass ofabout 62,483 daltons (SEQ ID NO:3). UspA2 is not a truncated orprocessed form of UspA1.

In a second embodiment, the present invention has identified thespecific epitope to which MAb 17C7 binds. A common peptide sequence,designated as the “3Q” peptide, found between amino acid residues480-502 and 582-604 of the UspA1 protein (SEQ ID NO:1 and residues355-377 of the UspA2 protein (SEQ ID NO:3) of M. catarrhalis strainO35E, encompasses the region which appears to be recognized by MAb 17C7.(Note that numbering of the amino acid residues is based upon strainO35E as provided in SEQ ID NO:3.) It is envisioned that this regionplays an important role in the biology of the pathogen and, from thisinformation, one will deduce amino acids residues that are critical inMAb 17C7 antibody binding. It also is envisioned that, based upon thisinformation, one will be able to design epitopic regions that haveeither a higher or lower affinity for the MAb 17C7 or other antibodies.Further embodiments of the present invention are discussed below.

In another preferred embodiment, the present invention provides DNAsegments, vectors and the like comprising at least one isolated gene,DNA segment or coding region that encodes a M. catarrhalis UspA1 orUspA2 protein, polypeptide, domain, peptide or any fusion proteinthereof. Herein are provided at least an isolated gene, DNA segment orcoding region that encodes a M. catarrhalis uspA1 gene comprising about2493 base pairs (bp) (SEQ ID NO:2) of strain O35E, about 3381 bp (SEQ IDNO:6) of strain 046E, about 3538 bp (SEQ ID NO:10) of strain TTA24, orabout 3292 bp (SEQ ID NO:14) of strain TTA37. Further provided are atleast an isolated gene, DNA segment or coding region that encodes a M.catarrhalis uspA2 gene comprising about 1728 bp (SEQ ID NO:4) of strainO35E, about 3295 bp (SEQ ID NO:8) of strain 046E, about 2673 bp (SEQ IDNO:12), or about 4228 bp (SEQ ID NO:16) of strain TTA37. It isenvisioned that the uspA1 and uspA2 genes will be useful in thepreparation of proteins, antibodies, screening assays for potentialcandidate drugs and the like to treat or inhibit, or even prevent, M.catarrhalis infections.

The present invention also provides for the use of the UspA1 or UspA2proteins or peptides as immunogenic carriers of other agents which areuseful for the treatment, inhibition or even prevention of otherbacterial, viral or parasitic infections. It is envisioned that eitherthe UspA1 or UspA2 antigen, or portions thereof, will be coupled,bonded, bound, conjugated or chemically-linked to one or more agents vialinkers, polylinkers or derivatized amino acids such that a bispecificor multivalent composition or vaccine which is useful for the treatment,inhibition or even prevention of infection by M. catarrhalis and anotherpathogen's) is prepared. It is further envisioned that the methods usedin the preparation of these compositions will be familiar to those ofskill in the art and, for example, similar to those used to prepareconjugates to keyhole limpet hemocyannin (KLH) or bovine serum albumin(BSA).

It is important to note that screening methods for diagnosis andprophylaxis are readily available, as set forth below. Thus, the abilityto (i) test peptides, mutant peptides and antibodies for theirreactivity with each other and (ii) test peptides and antibodies for theability to prevent infections in vivo, provide powerful tools to developclinically important reagents.

1.0 UspA Proteins, Peptides and Polypeptides

The present invention, in one embodiment, encompasses the two newprotein sequences, UspA1 and UspA2, and the peptide sequence AQQQDQH(SEQ ID NO:17) identified as the target epitope of MAb 17C7. Inaddition, inspection of the amino acid sequences of the UspA1 and UspA2proteins from four strains of M. catarrhalis indicated that each proteincontained at least one copy of the peptide YELAQQQDQH (SEQ ID NO:18)which binds Mab 17C7 or, in one instance, a peptide nearly identical andhaving the amino acid sequence YDLAQQQDQH (SEQ ID NO:19).

The peptide (YELAQQQDQH, SEQ ID NO:18) occurs twice in UspA1 from strainO35E at residues 486-495 and 588-597 (SEQ ID NO:1) and once in UspA2from strain O35E at residues 358-367 (SEQ ID NO:3). It occurs once inUspA1 from strain TTA24 at residues 497-506 (SEQ ID NO:9) and twice inUspA2 from strain TTA24 at residues 225-234 and 413-422 (SEQ ID NO:11).The peptide YDLAQQQDQH (SEQ ID NO:19) occurs once in UspA1 from strainO46E at residues 448-457 (SEQ ID NO:5) whereas the peptide YELAQQQDQH(SEQ ID NO:18) occurs once in this same protein at residues 649-658 (SEQID NO:5). The peptide YELAQQQDQH (SEQ ID NO:18) occurs once in UspA2from strain O46E at residues 416-425 (SEQ ID NO:7). The peptideYELAQQQDQH (SEQ ID NO:18) occurs twice in UspA1 from strain TTA37 atresidues 478-487 and 630-639 (SEQ ID NO:13) and twice in UspA2 fromstrain TTA37 at residues 522-531 and 681-690 (SEQ ID NO:15).

Also encompassed in the present invention are hybrid moleculescontaining portions from one UspA protein, for example the UspA1protein, fused with portions of the other UspA protein, in this examplethe UspA2 protein, or fused with other proteins which are useful foridentification, such as kanamycin-resistance, or other purposes in thescreening of potential vaccines or further characterization of the UspA1and UspA2 proteins. For example, one may fuse residues 1-350 of anyUspA1 with residues 351-576 of any UspA2. Alternatively, a fusion couldbe generated with sequences from three, four or even five peptideregions represented in a single UspA antigen. Also encompassed arefragments of the disclosed UspA1 and UspA2 molecules, as well asinsertion, deletion or replacement mutants in which non-UspA sequencesare introduced, UspA sequences are removed, or UspA sequences arereplaced with non-UspA sequences, respectively.

UspA1 and UspA2 proteins, according to the present invention, may beadvantageously cleaved into fragments for use in further structural orfunctional analysis, or in the generation of reagents such asUspA-related polypeptides and UspA-specific antibodies. This can beaccomplished by treating purified or unpurified UspA1 and/or UspA2 witha peptidase such as endoproteinase glu-C (Boehringer, Indianapolis,Ind.). Treatment with CNBr is another method by which UspA1 and/or UspA2fragments may be produced from their natural respective proteins.Recombinant techniques also can be used to produce specific fragments ofUspA1 or UspA2.

More subtle modifications and changes may be made in the structure ofthe encoded UspA1 or UspA2 polypeptides of the present invention andstill obtain a molecule that encodes a protein or peptide withcharacteristics of the natural UspA antigen. The following is adiscussion based upon changing the amino acids of a protein to create anequivalent, or even an improved, second-generation molecule. The aminoacid changes may be achieved by changing the codons of the DNA sequence,according to the following codon table:

TABLE I Amino acid names and abbreviations Codons Alanine Ala A GCA GCCGCG GCU Cysteine Cys C UGC UGU Aspartic acid Asp D GAC GAU Glutamic acidGlu E GAA GAG Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGUHistidine His H CAC CAU Isoleucine Ile I AUA AUC AUU Lysine Lys K AAAAAG Leucine Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUGAsparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln QCAA CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serine Ser S AGC AGU UCAUCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUUTryptophan Trp W UGG Tyrosine Tyr Y UAC UAU

It is known that certain amino acids may be substituted for other aminoacids in a protein structure in order to modify or improve its antigenicor immunogenic activity (see, e.g., Kyte & Doolittle, 1982; Hopp, U.S.Pat. No. 4,554,101, incorporated herein by reference). For example,through the substitution of alternative amino acids, smallconformational changes may be conferred upon a polypeptide which resultin increased activity or stability. Alternatively, amino acidsubstitutions in certain polypeptides may be utilized to provideresidues which may then be linked to other molecules to providepeptide-molecule conjugates which retain enough antigenicity of thestarting peptide to be useful for other purposes. For example, aselected UspA1 or UspA2 peptide bound to a solid support might beconstructed which would have particular advantages in diagnosticembodiments.

The importance of the hydropathic index of amino acids in conferringinteractive biological function on a protein has been discussedgenerally by Kyte & Doolittle (1982), wherein it is found that certainamino acids may be substituted for other amino acids having a similarhydropathic index or core and still retain a similar biologicalactivity. As displayed in Table II below, amino acids are assigned ahydropathic index on the basis of their hydrophobicity and chargecharacteristics. It is believed that the relative hydropathic characterof the amino acid determines the secondary structure of the resultantprotein, which in turn defines the interaction of the protein withsubstrate molecules. Preferred substitution which result in anantigenically equivalent peptide or protein will generally involve aminoacids having index scores within ±2 units of one another, and morepreferably within ±1 unit, and even more preferably, within ±0.5 units.

TABLE II Amino Acid Hydropathic Index Isoleucine 4.5 Valine 4.2 Leucine3.8 Phenylalanine 2.8 Cysteine/cystine 2.5 Methionine 1.9 Alanine 1.8Glycine −0.4 Threonine −0.7 Tryptophan −0.9 Serine −0.8 Tyrosine −1.3Proline −1.6 Histidine −3.2 Glutamic Acid −3.5 Glutamine −3.5 AsparticAcid −3.5 Asparagine −3.5 Lysine −3.9 Arginine −4.5

Thus, for example, isoleucine, which has a hydropathic index of +4.5,will preferably be exchanged with an amino acid such as valine (+4.2) orleucine (+3.8). Alternatively, at the other end of the scale, lysine(−3.9) will preferably be substituted for arginine (−4.5), and so on.

Substitution of like amino acids may also be made on the basis ofhydrophilicity, particularly where the biological functional equivalentprotein or peptide thereby created is intended for use in immunologicalembodiments. U.S. Pat. No. 4,554,101, incorporated herein by reference,states that the greatest local average hydrophilicity of a protein, asgoverned by the hydrophilicity of its adjacent amino acids, correlateswith its immunogenicity and antigenicity, i.e. with an importantbiological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, each amino acid has also beenassigned a hydrophilicity value. These values are detailed below inTable III.

TABLE III Amino Acid Hydrophilic Index arginine +3.0 lysine +3.0aspartate +3.0 ± 1 glutamate +3.0 ± 1 serine +0.3 asparagine +0.2glutamine +0.2 glycine   0 threonine −0.4 alanine −0.5 histidine −0.5proline −0.5 ± 1 cysteine −1.0 methionine −1.3 valine −1.5 leucine −1.8isoleucine −1.8 tyrosine −2.3 phenylalanine −2.5 tryptophan −3.4

It is understood that one amino acid can be substituted for anotherhaving a similar hydrophilicity value and still obtain a biologicallyequivalent, and in particular, an immunologically equivalent protein. Insuch changes, the substitution of amino acids whose hydrophilicityvalues are within ±2 is preferred, those which are within ±1 areparticularly preferred, and those within ±0.5 are even more particularlypreferred.

Accordingly, these amino acid substitutions are generally based on therelative similarity of R-group substituents, for example, in terms ofsize, electrophilic character, charge, and the like. In general,preferred substitutions which take various of the foregoingcharacteristics into consideration will be known to those of skill inthe art and include, for example, the following combinations: arginineand lysine; glutamate and aspartate; serine and threonine; glutamine andasparagine; and valine, leucine and isoleucine.

In addition, peptides derived from these polypeptides, includingpeptides of at least about 6 consecutive amino acids from thesesequences, are contemplated. Alternatively, such peptides may compriseabout 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59or 60 consecutive residues. For example, a peptide that comprises 6consecutive amino acid residues may comprise residues 1 to 6, 2 to 7, 3to 8 and so on of the UspA1 or UspA2 protein. Such peptides may berepresented by the formula

x to (x+n)=5′ to 3′ the positions of the first and last consecutiveresidues

where x is equal to any number from 1 to the full length of a UspA1 orUspA2 protein and n is equal to the length of the peptide minus 1. So,for UspA1, x=1 to 831, for UspA2, x=1 to 576. Where the peptide is 10residues long (n=10−1), the formula represents every 10-mer is possiblefor each antigen. For example, where x is equal to 1 the peptide wouldcomprise residues 1 to (1+[10−1]), or 1 to 10. Where x is equal to 2,the peptide would comprise residues 2 to (2+[10−2]), or 2 to 11, and soon.

Syntheses of peptides are readily achieved using conventional synthetictechniques such as the solid phase method (e.g., through the use of acommercially available peptide synthesizer such as an Applied BiosystemsModel 430A Peptide Synthesizer). Peptides synthesized in this manner maythen be aliquoted in predetermined amounts and stored in conventionalmanners, such as in aqueous solutions or, even more preferably, in apowder or lyophilized state pending use.

In general, due to the relative stability of peptides, they may bereadily stored in aqueous solutions for fairly long periods of time ifdesired, e.g., up to six months or more, in virtually any aqueoussolution without appreciable degradation or loss of antigenic activity.However, where extended aqueous storage is contemplated it willgenerally be desirable to include agents including buffers such as Trisor phosphate buffers to maintain a pH of 7.0 to 7.5. Moreover, it may bedesirable to include agents which will inhibit microbial growth, such assodium azide or Merthiolate. For extended storage in an aqueous state itwill be desirable to store the solutions at 4° C., or more preferably,frozen. Of course, where the peptide(s) are stored in a lyophilized orpowdered state, they may be stored virtually indefinitely, e.g., inmetered aliquots that may be rehydrated with a predetermined amount ofwater (preferably distilled, deionized) or buffer prior to use.

Of particular interest are peptides that represent epitopes that liewithin the UspA antigen and are encompassed by the UspA1 and UspA2proteins of the present invention. An “epitope” is a region of amolecule that stimulates a response from a T-cell or B-cell, and hence,elicits an immune response from these cells. An epitopic core sequence,as used herein, is a relatively short stretch of amino acids that isstructurally “complementary” to, and therefore will bind to, bindingsites on antibodies or T-cell receptors. It will be understood that, inthe context of the present disclosure, the term “complementary” refersto amino acids or peptides that exhibit an attractive force towards eachother. Thus, certain epitopic core sequences of the present inventionmay be operationally defined in terms of their ability to compete withor perhaps displace the binding of the corresponding UspA antigen to thecorresponding UspA-directed antisera.

The identification of epitopic core sequences is known to those of skillin the art. For example U.S. Pat. No. 4,554,101 teaches identificationand preparation of epitopes from amino acid sequences on the basis ofhydrophilicity, and by Chou-Fasman analyses. Numerous computer programsare available for use in predicting antigenic portions of proteins,examples of which include those programs based upon Jameson-Wolfanalyses (Jameson and Wolf, 1988; Wolf et al., 1988), the programPepPlot® (Brutlag et al., 1990; Weinberger et al., 1985), and other newprograms for protein tertiary structure prediction (Fetrow & Bryant,1993) that can be used in conjunction with computerized peptide sequenceanalysis programs.

In general, the size of the polypeptide antigen is not believed to beparticularly crucial, so long as it is at least large enough to carrythe identified core sequence or sequences. The smallest useful coresequence anticipated by the present disclosure would be on the order ofabout 6 amino acids in length. Thus, this size will generally correspondto the smallest peptide antigens prepared in accordance with theinvention. However, the size of the antigen may be larger where desired,so long as it contains a basic epitopic core sequence.

2.0 UspA1 and UspA2 Nucleic Acids

In addition to polypeptides, the present invention also encompassesnucleic acids encoding the UspA1 (SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:10and SEQ ID NO:14) and UspA2 (SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:12 andSEQ ID NO:16) proteins from the exemplary M. catarrhalis strains O35E,O46E, TTA24 and TTA37, respectively. Because of the degeneracy of thegenetic code, many other nucleic acids also may encode a given UspA1 orUspA2 protein. For example, four different three-base codons encode theamino acids alanine, glycine, proline, threonine and valine, while sixdifferent codons encode arginine, leucine and serine. Only methionineand tryptophan are encoded by a single codon. Table I provides a list ofamino acids and their corresponding codons for use in such embodiments.In order to generate any nucleic acid encoding UspA1 or UspA2, one needonly refer to the codon table provided herein. Substitution of thenatural codon with any codon encoding the same amino acid will result ina distinct nucleic acid that encodes UspA1 or UspA2. As a practicalmatter, this can be accomplished by site-directed mutagenesis of anexisting uspA1 or uspA2 gene or de novo chemical synthesis of one ormore nucleic acids.

These observations regarding codon selection, site-directed mutagenesisand chemical synthesis apply with equal force to the discussion ofsubstitutional mutant UspA1 or UspA2 peptides and polypeptides, as setforth above. More specifically, substitutional mutants generated bysite-directed changes in the nucleic acid sequence that are designed toalter one or more codons of a given polypeptide or epitope may provide amore convenient way of generating large numbers of mutants in a rapidfashion. The nucleic acids of the present invention provide for a simpleway to generate fragments (e.g., truncations) of UspA1 or UspA2,UspA1-UspA2 fusion molecules (discussed above) and UspA1 or UspA2fusions with other molecules. For example, utilization of restrictionenzymes and nuclease in the uspA1 or uspA2 gene permits one tomanipulate the structure of these genes, and the resulting geneproducts.

The nucleic acid sequence information provided by the present disclosurealso allows for the preparation of relatively short DNA (or RNA)sequences that have the ability to specifically hybridize to genesequences of the selected uspA1 or uspA2 gene. In these aspects nucleicacid probes of an appropriate length are prepared based on aconsideration of the coding sequence of the uspA1 or uspA2 gene, orflanking regions near the uspA1 or uspA2 gene, such as regionsdownstream and upstream in the M. catarrhalis chromosome. The ability ofsuch nucleic acid probes to specifically hybridize to either uspA1 oruspA2 gene sequences lends them particular utility in a variety ofembodiments. For example, the probes can be used in a variety ofdiagnostic assays for detecting the presence of pathogenic organisms ina given sample. In addition, these oligonucleotides can be inserted, inframe, into expression constructs for the purpose of screening thecorresponding peptides for reactivity with existing antibodies or forthe ability to generate diagnostic or therapeutic reagents.

To provide certain of the advantages in accordance with the invention,the preferred nucleic acid sequence employed for hybridization studiesor assays includes sequences that are complementary to at least a 10 to20, or so, nucleotide stretch of the sequence, although sequences of 30to 60 or so nucleotides are also envisioned to be useful. A size of atleast 9 nucleotides in length helps to ensure that the fragment will beof sufficient length to form a duplex molecule that is both stable andselective. Though molecules having complementary sequences overstretches greater than 10 bases in length are generally preferred, inorder to increase stability and selectivity of the hybrid, and therebyimprove the quality and degree of the specific hybrid moleculesobtained. Thus, one will generally prefer to design nucleic acidmolecules having either uspA1 or uspA2 gene-complementary stretches of15 to 20 nucleotides, or even longer, such as 30 to 60, where desired.Such fragments may be readily prepared by, for example, directlysynthesizing the fragment by chemical means, by application of nucleicacid reproduction technology, such as the PCR™ technology of U.S. Pat.No. 4,603,102, or by introducing selected sequences into recombinantvectors for recombinant production.

The probes that would be useful may be derived from any portion of thesequences of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 orSEQ ID NO:10 or SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16. Therefore,probes are specifically contemplated that comprise nucleotides 1 to 9,or 2 to 10, or 3 to 11 and so forth up to a probe comprising the last 9nucleotides of the nucleotide sequence of SEQ ID NO:2 or SEQ ID NO:4 orSEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO:12 or SEQ IDNO:14 or SEQ ID NO:16. Thus, each probe would comprise at least about 9linear nucleotides of the nucleotide sequence of SEQ ID NO:2 or SEQ IDNO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO:12 orSEQ ID NO:14 or SEQ ID NO:16, designated by the formula “n to n+8,”where n is an integer from 1 to the number of nucleotides in thesequence. Longer probes that hybridize to the uspA1 or uspA2 gene underlow, medium, medium-high and high stringency conditions are alsocontemplated, including those that comprise the entire nucleotidesequence of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 orSEQ ID NO:IO or SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16. Thishypothetical may be repeated for probes having lengths of about 10, 11,12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80,90, 100 and greater bases.

In that the UspA antigenic epitopes of the present invention arebelieved to be indicative of pathogenic Moraxella species as exemplifiedby strains O35E, O46E, TTA24 and TTA37, the probes of the presentinvention will find particular utility as the basis for diagnostichybridization assays for detecting UspA1 or UspA2 DNA in clinicalsamples. Exemplary clinical samples that can be used in the diagnosis ofinfections are thus any samples which could possibly include Moraxellanucleic acid, including middle ear fluid, sputum, mucus, bronchoalveolarfluid, amniotic fluid or the like. A variety of hybridization techniquesand systems are known which can be used in connection with thehybridization aspects of the invention, including diagnostic assays suchas those described in Falkow et al., U.S. Pat. No. 4,358,535. Dependingon the application envisioned, one will desire to employ varyingconditions of hybridization to achieve varying degrees of selectivity ofthe probe toward the target sequence. For applications requiring a highdegree of selectivity, one will typically desire to employ relativelystringent conditions to form the hybrids, for example, one will selectrelatively low salt and/or high temperature conditions, such as providedby 0.02M-0.15M NaCl at temperatures of 50° C. to 70° C. These conditionsare particularly selective, and tolerate little, if any, mismatchbetween the probe and the template or target strand.

Of course, for some applications, for example, where one desires toprepare mutants employing a mutant primer strand hybridized to anunderlying template, less stringent hybridization conditions are calledfor in order to allow formation of the heteroduplex. In thesecircumstances, one would desire to employ conditions such as 0.15M-0.9Msalt, at temperatures ranging from 20° C. to 55° C. In any case, it isgenerally appreciated that conditions can be rendered more stringent bythe addition of increasing amounts of formarnide, which serves todestabilize the hybrid duplex in the same manner as increasedtemperature. Thus, hybridization conditions can be readily manipulated,and the method of choice will generally depend on the desired results.

In certain embodiments, one may desire to employ nucleic acid probes toisolate variants from clone banks containing mutated clones. Inparticular embodiments, mutant clone colonies growing on solid mediawhich contain variants of the UspA1 and/or UspA2 sequence could beidentified on duplicate filters using hybridization conditions andmethods, such as those used in colony blot assays, to obtainhybridization only between probes containing sequence variants andnucleic acid sequence variants contained in specific colonies. In thismanner, small hybridization probes containing short variant sequences ofeither the uspA1 or uspA2 gene may be utilized to identify those clonesgrowing on solid media which contain sequence variants of the entireuspA1 or uspA2 gene. These clones can then be grown to obtain desiredquantities of the variant UspA1 or UspA2 nucleic acid sequences or thecorresponding UspA antigen.

In clinical diagnostic embodiments, nucleic acid sequences of thepresent invention are used in combination with an appropriate means,such as a label, for determining hybridization. A wide variety ofappropriate indicator means are known in the art, including radioactive,enzymatic or other ligands, such as avidin/biotin, which are capable ofgiving a detectable signal. In preferred diagnostic embodiments, onewill likely desire to employ an enzyme tag such as urease, alkalinephosphatase or peroxidase, instead of radioactive or other environmentalundesirable reagents. In the case of enzyme tags, colorimetric indicatorsubstrates are known which can be employed to provide a means visible tothe human eye or spectrophotometrically, to identify specifichybridization with pathogen nucleic acid-containing samples.

In general, it is envisioned that the hybridization probes describedherein will be useful both as reagents in solution hybridizations aswell as in embodiments employing a solid phase. In embodiments involvinga solid phase, the test DNA (or RNA) from suspected clinical samples,such as exudates, body fluids (e.g., amniotic fluid, middle eareffusion, bronchoalveolar lavage fluid) or even tissues, is adsorbed orotherwise affixed to a selected matrix or surface. This fixed,single-stranded nucleic acid is then subjected to specific hybridizationwith selected probes under desired conditions. The selected conditionswill depend on the particular circumstances based on the particularcriteria required (depending, for example, on the G+C contents, type oftarget nucleic acid, source of nucleic acid, size of hybridizationprobe, etc.). Following washing of the hybridized surface so as toremove nonspecifically bound probe molecules, specific hybridization isdetected, or even quantified, by means of the label.

The nucleic acid sequences which encode for the UspA1 and/or UspA2epitopes, or their variants, may be useful in conjunction with PCR™methodology to detect M. catarrhalis. In general, by applying the PCR™technology as set out, e.g., in U.S. Pat. No. 4,603,102, one may utilizevarious portions of either the uspA1 or uspA2 sequence asoligonucleotide probes for the PCR™ amplification of a defined portionof a uspA1 or uspA2 nucleic acid in a sample. The amplified portion ofthe uspA1 or uspA2 sequence may then be detected by hybridization with ahybridization probe containing a complementary sequence. In this manner,extremely small concentrations of M. catarrhalis nucleic acid maydetected in a sample utilizing uspA1 or uspA2 sequences.

3.0 Vectors, Host Cells and Cultures for Producing UspA1 and/or UspA2Antigens

In order to express a UspA1 and/or UspA2 polypeptide, it is necessary toprovide an uspA1 and/or uspA2 gene in an expression cassette. Theexpression cassette contains a UspA1 and/or UspA2-encoding nucleic acidunder transcriptional control of a promoter. A “promoter” refers to aDNA sequence recognized by the synthetic machinery of the cell, orintroduced synthetic machinery, required to initiate the specifictranscription of a gene. The phrase “under transcriptional control”means that the promoter is in the correct location and orientation inrelation to the nucleic acid to control RNA polymerase initiation andexpression of the gene. Those promoters most commonly used inprokaryotic recombinant DNA construction include the B-lactamase(penicillinase) and lactose promoter systems (Chang et al., 1978;Itakura et al., 1977; Goeddel et al., 1979) and a tryptophan (trp)promoter system (Goeddel et al., 1980; EPO Appl. Publ. No. 0036776).While these are the most commonly used, other microbial promoters havebeen discovered and utilized, and details concerning their nucleotidesequences have been published, enabling a skilled worker to ligate themfunctionally with plasmid vectors (EPO Appl. Publ. No. 0036776).Additional examples of useful promoters are provided in Table IV below.

TABLE IV Promoters References Immunoglobulin Hanerji et al., 1983;Gilles et al., 1983; Heavy Chain Grosschedl and Baltimore, 1985;Atchinson and Perry, 1986, 1987; Imler et al., 1987; Weinberger et al.,1988; Kiledjian et al., 1988; Porton et al., 1990 Immunoglobulin Queenand Baltimore, 1983; Picard Light Chain and Schaffner, 1984 T-CellReceptor Luria et al., 1987, Winoto and Baltimore, 1989; Redondo et al.,1990 HLA DQ a and Sullivan and Peterlin, 1987 DQ β β-InterferonGoodbourn et al., 1986; Fujita et al., 1987; Goodbourn and Maniatis,1985 Interleukin-2 Greene et al., 1989 Interleukin-2 Greene et al.,1989; Lin et al., 1990 Receptor MHC Class II 5 Koch et al., 1989 MHCClass II Sherman et al., 1989 HLA-DRa β-Actin Kawamoto et al., 1988; Nget al., 1989 Muscle Creatine Jaynes et al., 1988; Horlick and Benfield,1989; Kinase Johnson et al., 1989a Prealbumin Costa et al., 1988(Transthyretin) Elastase I Omitz et al., 1987 Metallothionein Karin etal., 1987; Culotta and Hamer, 1989 Collagenase Pinkert et al., 1987;Angel et al., 1987 Albumin Gene Pinkert et al., 1987, Tronche et al.,1989, 1990 a-Fetoprotein Godbout et al., 1988; Campere and Tilghman,1989 t-Globin Bodine and Ley, 1987; Perez-Stable and Constantini, 1990β-Globin Trudel and Constantini, 1987 e-fos Cohen et al., 1987 c-HA-rasTriesman, 1986; Deschamps et al., 1985 Insulin Edlund et al., 1985Neural Cell Hirsch et al., 1990 Adhesion Molecule (NCAM)a_(1-Antitrypain) Latimer et al., 1990 H2B (TH2B) Hwang et al., 1990Histone Mouse or Type Ripe et al., 1989 I Collagen Glucose-RegulatedChang et al., 1989 Proteins (GRP94 and GRP78) Rat Growth Larsen et al.,1986 Hormone Human Serum Edbrooke et al., 1989 Amyloid A (SAA) TroponinI (TN I) Yutzey et al., 1989 Platelet-Derived Pech et al., 1989 GrowthFactor Duchenne Muscular Klamut et al., 1990 Dystrophy SV40 Banerji etal., 1981; Moreau et al., 1981; Sleigh Lockett, 1985; Firak andSubramanian, 1986; Herr and Clarke, 1986; Imbra and Karin, 1986; Kadeschand Berg, 1986; Wang and Calame, 1986; Ondek et al., 1987; Kuhl et al.,1987 Schaffner et al., 1988 Polyoma Swartzendruber and Lehman, 1975;Vasseur et al., 1980; Katinka et al., 1980, 1981; Tyndell et al., 1981;Dandolo et al., 1983; deVilliers et al., 1984; Hen et al., 1986; Satakeet al., 1988; Campbell and Villarreal, 1988 Retroviruses Kriegler andBotchan, 1982, 1983; Levinson et al., 1982; Kriegler et al., 1983,1984a,b, 1988; Bosze et al., 1986; Miksicek et al., 1986; Celander andHaseltine, 1987; Thiesen et al., 1988; Celander et al., 1988; Chol etal., 1988; Reisman and Rotter, 1989 Papilloma Virus Campo et al., 1983;Lusky et al., 1983; Spandidos and Wilkie, 1983; Spalholz et al., 1985;Lusky and Botchan, 1986; Cripe et al., 1987; Gloss et al., 1987;Hirochika et al., 1987, Stephens and Hentschel, 1987; Glue et al., 1988Hepatitis B Virus Bulla and Siddiqui, 1986; Jameel and Siddiqui, 1986;Shaul and Ben-Levy, 1987; Spandau and Lee, 1988; Vannice and Levinson,1988 Human Muesing et al., 1987; Hauber and Cullan, 1988;Immunodeficiency Jakobovits et al., 1988; Feng and Holland, Virus 1988;Takebe et al., 1988; Rowen et al., 1988; Berkhout et al., 1989; Laspiaet al., 1989; Sharp and Marciniak, 1989; Braddock et al., 1989Cytomegalovirus Weber et al., 1984; Boshart et al., 1985; Foecking andHofstetter, 1986 Gibbon Ape Holbrook et al., 1987; Quinn et al., 1989Leukemia Virus

The appropriate expression cassette can be inserted into a commerciallyavailable expression vector by standard subcloning techniques. Forexample, the E. coli vectors pUC or pBluescript# may be used accordingto the present invention to produce recombinant UspA1 and/or UspA2polypeptide in vitro. The manipulation of these vectors is well known inthe art. In general, plasmid vectors containing replicon and controlsequences which are derived from species compatible with the host cellare used in connection with these hosts. The vector ordinarily carries areplication site, as well as marking sequences which are capable ofproviding phenotypic selection in transformed cells. For example, E.coli is typically transformed using pBR322, a plasmid derived from an E.coli species (Bolivar et al., 1977). pBR322 contains genes forampicillin and tetracycline resistance and thus provides easy means foridentifying transformed cells. The pBR plasmid, or other microbialplasmid or phage must also contain, or be modified to contain, promoterswhich can be used by the microbial organism for expression of its ownproteins.

In addition, phage vectors containing replicon and control sequencesthat are compatible with the host microorganism can be used as atransforming vector in connection with these hosts. For example, thephage lambda GEM™-11 may be utilized in making recombinant phage vectorwhich can be used to transform host cells, such as E. coli LE392.

In one embodiment, the UspA antigen is expressed as a fusion protein byusing the pGEX4T-2 protein fusion system (Pharmacia LKB, Piscataway,N.J.), allowing characterization of the UspA antigen as comprising boththe UspA1 and UspA2 proteins. Additional examples of fusion proteinexpression systems are the glutathione S-transferase system (Pharmacia,Piscataway, N.J.), the maltose binding protein system (NEB, Beverley,Mass.), the FLAG system (IBI, New Haven, Conn.), and the 6×His system(Qiagen, Chatsworth, Calif.). Some of these fusion systems producerecombinant protein bearing only a small number of additional aminoacids, which are unlikely to affect the functional capacity of therecombinant protein. For example, both the FLAG system and the 6×Hissystem add only short sequences, both of which are known to be poorlyantigenic and which do not adversely affect folding of the protein toits native conformation. Other fusion systems produce proteins where itis desirable to excise the fusion partner from the desired protein. Inanother embodiment, the fusion partner is linked to the recombinantprotein by a peptide sequence containing a specific recognition sequencefor a protease. Examples of suitable sequences are those recognized bythe Tobacco Etch Virus protease (Life Technologies, Gaithersburg, Md.)or Factor Xa (New England Biolabs, Beverley, Mass.).

E. coli is a preferred prokaryotic host. For example, E. coli strain RR1is particularly useful. Other microbial strains which may be usedinclude E. coli strains such as E. coli LE392, E. coli B, and E. coli X1776 (ATCC No. 31537). The aforementioned strains, as well as E. coliW3110 (F-, lambda-, prototrophic, ATCC No. 273325), bacilli such asBacillus subtilis, or other enterobacteriaceae such as Salmonellatyphimurium or Serratia marcescens, and various Pseudomonas species maybe used. These examples are, of course, intended to be illustrativerather than limiting. Recombinant bacterial cells, for example E. coli,are grown in any of a number of suitable media, for example LB, and theexpression of the recombinant polypeptide induced by adding IPTG to themedia or switching incubation to a higher temperature. After culturingthe bacteria for a further period of between 2 and 24 hours, the cellsare collected by centrifugation and washed to remove residual media. Thebacterial cells are then lysed, for example, by disruption in a cellhomogenizer and centrifuged to separate the dense inclusion bodies andcell membranes from the soluble cell components. This centrifugation canbe performed under conditions whereby the dense inclusion bodies areselectively enriched by incorporation of sugars such as sucrose into thebuffer and centrifugation at a selective speed.

If the recombinant protein is expressed in the inclusion bodies, as isthe case in many instances, these can be washed in any of severalsolutions to remove some of the contaminating host proteins, thensolubilized in solutions containing high concentrations of urea (e.g.8M) or chaotropic agents such as guanidine hydrochloride in the presenceof reducing agents such as β-mercaptoethanol or DTT (dithiothreitol).

Under some circumstances, it may be advantageous to incubate thepolypeptide for several hours under conditions suitable for the proteinto undergo a refolding process into a conformation which more closelyresembles that of the native protein. Such conditions generally includelow protein concentrations less than 500 μg/ml, low levels of reducingagent, concentrations of urea less than 2 M and often the presence ofreagents such as a mixture of reduced and oxidized glutathione whichfacilitate the interchange of disulfide bonds within the proteinmolecule.

The refolding process can be monitored, for example, by SDS-PAGE or withantibodies which are specific for the native molecule (which can beobtained from animals vaccinated with the native molecule isolated frombacteria). Following refolding, the protein can then be purified furtherand separated from the refolding mixture by chromatography on any ofseveral supports including ion exchange resins, gel permeation resins oron a variety of affinity columns.

There are a variety of other eukaryotic vectors that provide a suitablevehicle in which recombinant UspA proteins can be produced. In variousembodiments of the invention, the expression construct may comprise avirus or engineered construct derived from a viral genome. The abilityof certain viruses to enter cells via receptor-mediated endocytosis andto integrate into host cell genome and express viral genes stably andefficiently have made them attractive candidates for the transfer offoreign genes into mammalian cells (Ridgeway, 1988; Nicolas andRubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986). The firstviruses used as vectors were DNA viruses including the papovaviruses(simian virus 40 (SV40), bovine papilloma virus, and polyoma) (Ridgeway,1988; Baichwal and Sugden, 1986) and adenoviruses (Ridgeway, 1988;Baichwal and Sugden, 1986) and adeno-associated viruses. Retrovirusesalso are attractive gene transfer vehicles (Nicolas and Rubenstein,1988; Temin, 1986) as are vaccina virus (Ridgewvay, 1988)adeno-associated virus (Ridgeway, 1988) and herpes simplex virus (HSV)(Glorioso et al., 1995). Such vectors may be used to (i) transform celllines in vitro for the purpose of expressing proteins of interest or(ii) to transform cells in vitro or in vivo to provide therapeuticpolypeptides in a gene therapy scenario.

With respect to eukaryotic vectors, the term promoter will be used hereto refer to a group of transcriptional control modules that areclustered around the initiation site for RNA polymerase II. Much of thethinking about how promoters are organized derives from analyses ofseveral viral promoters, including those for the HSV thymidine kinase(tk) and SV40 early transcription units. These studies, augmented bymore recent work, have shown that promoters are composed of discretefunctional modules, each consisting of approximately 7-20 bp of DNA, andcontaining one or more recognition sites for transcriptional activatoror repressor proteins.

At least one module in each promoter functions to position the startsite for RNA synthesis. The best known example of this is the TATA box,but in some promoters lacking a TATA box, such as the promoter for themammalian terminal deoxynucleotidyl transferase gene and the promoterfor the SV40 late genes, a discrete element overlying the start siteitself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 bpupstream of the start site, although a number of promoters have recentlybeen shown to contain functional elements downstream of the start siteas well. The spacing between promoter elements frequently is flexible,so that promoter function is preserved when elements are inverted ormoved relative to one another. In the tk promoter, the spacing betweenpromoter elements can be increased to 50 bp apart before activity beginsto decline. Depending on the promoter, it appears that individualelements can function either cooperatively or independently to activatetranscription.

The particular promoter that is employed to control the expression of anucleic acid is not believed to be critical, so long as it is capable ofexpressing the nucleic acid in the targeted cell. Thus, where a humancell is targeted, it is preferable to position the nucleic acid codingregion adjacent to and under the control of a promoter that is capableof being expressed in a human cell. Generally speaking, such a promotermight include either a human or viral promoter. Preferred promotersinclude those derived from HSV, including the α4 promoter. Anotherpreferred embodiment is the tetracycline controlled promoter.

In various other embodiments, the human cytomegalovirus (CMV) immediateearly gene promoter, the SV40 early promoter and the Rous sarcoma viruslong terminal repeat can be used to obtain high-level expression oftransgenes. The use of other viral or mammalian cellular or bacterialphage promoters which are well-known in the art to achieve expression ofa transgene is contemplated as well, provided that the levels ofexpression are sufficient for a given purpose. Table IV lists severalpromoters which may be employed, in the context of the presentinvention, to regulate the expression of a transgene. This list is notintended to be exhaustive of all the possible elements involved in thepromotion of transgene expression but, merely, to be exemplary thereof.

Enhancers were originally detected as genetic elements that increasedtranscription from a promoter located at a distant position on the samemolecule of DNA. This ability to act over a large distance had littleprecedent in classic studies of prokaryotic transcriptional regulation.Subsequent work showed that regions of DNA with enhancer activity areorganized much like promoters. That is, they are composed of manyindividual elements, each of which binds to one or more transcriptionalproteins.

The basic distinction between enhancers and promoters is operational. Anenhancer region as a whole must be able to stimulate transcription at adistance; this need not be true of a promoter region or its componentelements. On the other hand, a promoter must have one or more elementsthat direct initiation of RNA synthesis at a particular site and in aparticular orientation, whereas enhancers lack these specificities.Promoters and enhancers are often overlapping and contiguous, oftenseeming to have a very similar modular organization. Table V listsseveral enhancers, of course, this list is not meant to be limiting butexemplary.

TABLE V Enhancer Inducer References MT II Phorbol Palmiter et al., 1982;Haslinger and Ester (TFA) Karin, 1985; Searle et al., 1985; Stuart Heavyet al., 1985; Imagawa et al., 1987; metals Karin ®, 1987; Angel et al.,1987b; McNeall et al., 1989 MMTV Gluco- Huang et al., 1981; Lee et al.,1981; (mouse corticoids Majors and Varmus, 1983; Chandler mammary etal., 1983; Lee et al., 1984; Fonta et tumor al., 1985; Sakai et al.,1986 virus) β-Interferon poly(rl)X Tavernier et al., 1983 poly(rc)Adenovirus Ela Imperiale and Nevins, 1984 5 E2 Collagenase Phorbol Angleet al., 1987a Ester (TPA) Stromelysin Phorbol Angle et al., 1987b Ester(TPA) SV40 Phorbol Angel et al., 1987b Ester (TPA) Murine Interferon, MXGene Newcastle Disease Virus GRP78 Gene A23178 Resendez et al., 1988a-2-Macro- IL-6 Kunz et al., 1989 globulin Vimentin Serum Rittling etal., 1989 MHC Class Interferon Blanar et al., 1989 I Gene H-2kb HSP70Ela, SV40 Taylor et al., 1989; Taylor and Large T Kingston, Antigen1990a,b Proliferin Phorbol Mordacq and Linzer, 1989 Ester-TPA Tumor FMAHensel et al., 1989 Necrosis Factor Thyroid Thyroid Chatterjee et al.,1989 Hormone Stimulating Hormone a Gene

Additionally any promoter/enhancer combination (as per the EukaryoticPromoter Data Base EPDB) could also be used to drive expression of atransgene. Use of a T3, T7 or SP6 cytoplasmic expression system isanother possible embodiment. Eukaryotic cells can support cytoplasmictranscription from certain bacterial promoters if the appropriatebacterial polymerase is provided, either as part of the delivery complexor as an additional genetic expression construct.

Host cells include eukaryotic microbes, such as yeast cultures may alsobe used. Saccharomyces cerevisiae, or common bakers yeast is the mostcommonly used among eukaryotic microorganisms, although a number ofother strains are commonly available. For expression in Saccharomyces,the plasmid YRp7, for example, is commonly used (Stinchcomb et al.,1979; Kingsman et al., 1979; Tschemper et al., 1980). This plasmidalready contains the trpl gene which provides a selection marker for amutant strain of yeast lacking the ability to grow in tryptophan, forexample ATCC No. 44076 or PEP4-1 (Jones, 1977). The presence of the trpllesion as a characteristic of the yeast host cell genome then providesan effective environment for detecting transformation by growth in theabsence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters for3-phosphoglycerate kinase (Hitzeman et al., 1980) or other glycolyticenzymes (Hess et al., 1968; Holland et al., 1978), such as enolase,glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvatedecarboxylase, phosphofructokinase, glucose-6-phosphate isomerase,3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase,phosphoglucose isomerase, and glucokinase. In constructing suitableexpression plasmids, the termination sequences associated with thesegenes are also ligated into the expression vector 3′ of the sequencedesired to be expressed to provide polyadenylation of the mRNA andtermination. Other promoters, which have the additional advantage oftranscription controlled by growth conditions are the promoter regionfor alcohol dehydrogenase 2, isocytochrome C, acid phosphatase,degradative enzymes associated with nitrogen metabolism, and theaforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymesresponsible for maltose and galactose utilization. Any plasmid vectorcontaining a yeast-compatible promoter, origin of replication andtermination sequences is suitable.

In addition to eukaryotic microorganisms, cultures of cells derived frommulticellular organisms may also be used as hosts. In principle, anysuch cell culture is workable, whether from vertebrate or invertebrateculture. However, interest has been greatest in vertebrate cells, andpropagation of vertebrate cells in culture (tissue culture) has become aroutine procedure in recent years (Tissue Culture, 1973). Examples ofsuch useful host cell lines are VERO and HeLa cells, Chinese hamsterovary (CHO) cell lines, and W138, BHK, COS-7, 293 and MDCK. cell lines.Expression vectors for such cells ordinarily include (if necessary) anorigin of replication, a promoter located in front of the gene to beexpressed, along with any necessary ribosome binding sites, RNA splicesites, polyadenylation site, and transcriptional terminator sequences.

4.0 Preparation of Antibodies to UspA Proteins

Antibodies to UspA1 or UspA2 peptides or polypeptides may be readilyprepared through use of well-known techniques, such as those exemplifiedin U.S. Pat. No. 4,196,265. Typically, this technique involvesimmunizing a suitable animal with a selected immunogen composition,e.g., purified or partially purified protein, synthetic protein orfragments thereof, as discussed in the section on vaccines. Animals tobe immunized are mammals such as cats, dogs and horses, although thereis no limitation other than that the subject be capable of mounting animmune response of some kind. The immunizing composition is administeredin a manner effective to stimulate antibody producing cells. Rodentssuch as mice and rats are preferred animals, however, the use of rabbit,sheep or frog cells is possible. The use of rats may provide certainadvantages, but mice are preferred, with the BALB/c mouse being mostpreferred as the most routinely used animal and one that generally givesa higher percentage of stable fusions.

For generation of monoclonal antibodies (MAbs), following immunization,somatic cells with the potential for producing antibodies, specificallyB lymphocytes (B cells), are selected for use in the MAb generatingprotocol. These cells may be obtained from biopsies spleens, tonsils orlymph nodes, or from a peripheral blood sample. Spleen cells andperipheral blood cells are preferred, the former because they are a richsource of antibody-producing cells that are in the dividing plasmablaststage, and the latter because peripheral blood is easily accessible.Often, a panel of animals will have been immunized and the spleen of theanimal with the highest antibody titer removed. Spleen lymphocytes areobtained by homogenizing the spleen with a syringe. Typically, a spleenfrom an immunized mouse contains approximately 5×10⁷ to 2×10⁸lymphocytes.

The antibody-producing B cells from the immunized animal are then fusedwith cells of an immortal myeloma cell line, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma-producing fusion procedures preferably arenon-antibody-producing, have high fusion efficiency and enzymedeficiencies that render them incapable of growing in certain selectivemedia which support the growth of only the desired fused cells, called“hybridomas.”

Any one of a number of myeloma cells may be used and these are known tothose of skill in the art. For example, where the immunized animal is amouse, one may use P3-X63/Ag8, X63-Ag8.653, NS1/1.Ag 4 1, Sp210-Ag14,FO, NSO/U, MPC-11, MPC11-X45-GTG 1.7 and S194/5XX0 Bul; for rats, onemay use R210.RCY3, Y3-Ag 1.2.3, IR983F and 4B210; and U-266,GM1500-GRG2, LICR-LON-HMy2 and UC729-6 are all useful in connection withhuman cell fusions.

One preferred murine myeloma cell line is the NS-1 myeloma cell line(also tenned P3-NS-1-Ag4-1), which is readily available from the NIGMSHuman Genetic Mutant Cell Repository by requesting cell line repositorynumber GM3573. Another mouse myeloma cell line that may be used is the8-azaguanine-resistant mouse murine myeloma SP2/0 non-producer cellline.

Methods for generating hybrids of antibody-producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 proportion, though the proportion may vary fromabout 20:1 to about 1:1, respectively, in the presence of an agent oragents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler & Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al. (1977). The use cfelectrically induced fusion methods is also appropriate.

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. This does not pose a problem, however, as theviable, fused hybrids are differentiated from the parental, unfusedcells particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culture in a selective medium. Theselective medium generally is one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby single-clone dilution in microtiter plates, followed by testing theindividual clonal supernatants (after about two to three weeks) for thedesired reactivity. The assay should be sensitive, simple and rapid,such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays,plaque assays, dot immunobinding assays, and the like.

The selected hybridomas are then serially diluted and cloned intoindividual antibody-producing cell lines, which clones can then bepropagated indefinitely to provide MAbs. The cell lines may be exploitedfor MAb production in two basic ways. A sample of the hybridoma can beinjected, usually in the peritoneal cavity, into a histocompatibleanimal of the type that was used to provide the somatic and myelomacells for the original fusion. The injected animal develops tumorssecreting the specific monoclonal antibody produced by the fused cellhybrid. The body fluids of the animal, such as serum or ascites fluid,can then be tapped to provide MAbs in high concentration. The individualcell lines could also be cultured in vitro, where the MAbs are naturallysecreted into the culture medium from which they can be readily obtainedin high concentrations. MAbs produced by either means may be furtherpurified, if desired, using filtration, centrifugation and variouschromatographic methods such as HPLC or affinity chromatography.Monoclonal antibodies of the present invention also includeanti-idiotypic antibodies produced by methods well-known in the art.Monoclonal antibodies according to the present invention also may bemonoclonal heteroconjugates, i.e., hybrids of two or more antibodymolecules. In another embodiment, monoclonal antibodies according to theinvention are chimeric monoclonal antibodies. In one approach, thechimeric monoclonal antibody is engineered by cloning recombinant DNAcontaining the promoter, leader, and variable-region sequences from amouse antibody producing cell and the constant-region exons from a humanantibody gene. The antibody encoded by such a recombinant gene is amouse-human chimera. Its antibody specificity is determined by thevariable region derived from mouse sequences. Its isotype, which isdetermined by the constant region, is derived from human DNA.

In another embodiment, the monoclonal antibody according to the presentinvention is a “humanized” monoclonal antibody, produced by techniqueswell-known in the art. That is, mouse complementary determining regions(“CDRs”) are transferred from heavy and light V-chains of the mouse Iginto a human V-domain, followed by the replacement of some humanresidues in the framework regions of their murine counterparts.“Humanized” monoclonal antibodies in accordance with this invention areespecially suitable for use in in vivo diagnostic and therapeuticmethods for treating Moraxella infections.

As stated above, the monoclonal antibodies and fragments thereofaccording to this invention can be multiplied according to in vitro andin vivo methods well-known in the art. Multiplication in vitro iscarried out in suitable culture media such as Dulbecco's modified Eaglemedium or RPMI 1640 medium, optionally replenished by a mammalian serumsuch as fetal calf serum or trace elements and growth-sustainingsupplements, e.g., feeder cells, such as normal mouse peritoneal exudatecells, spleen cells, bone marrow macrophages or the like. In vitroproduction provides relatively pure antibody preparations and allowsscale-up to give large amounts of the desired antibodies. Techniques forlarge scale hybridoma cultivation under tissue culture conditions areknown in the art and include homogenous suspension culture, e.g., in anairlift reactor or in a continuous stirrer reactor or immobilized orentrapped cell culture.

Large amounts of the monoclonal antibody of the present invention alsomay be obtained by multiplying hybridoma cells in vivo. Cell clones areinjected into mammals which are histocompatible with the parent cells,e.g., syngeneic mice, to cause growth of antibody-producing tumors.Optionally, the animals are primed with a hydrocarbon, especially oilssuch as Pristane (tetramethylpentadecane) prior to injection.

In accordance with the present invention, fragments of the monoclonalantibody of the invention can be obtained from monoclonal antibodiesproduced as described above, by methods which include digestion withenzymes such as pepsin or papain and/or cleavage of disulfide bonds bychemical reduction. Alternatively, monoclonal antibody fragmentsencompassed by the present invention can be synthesized using anautomated peptide synthesizer, or they may be produced manually usingtechniques well known in the art.

The monoclonal conjugates of the present invention are prepared bymethods known in the art, e.g., by reacting a monoclonal antibodyprepared as described above with, for instance, an enzyme in thepresence of a coupling agent such as glutaraldehyde or periodate.Conjugates with fluorescein markers are prepared in the presence ofthese coupling agents, or by reaction with an isothiocyanate. Conjugateswith metal chelates are similarly produced. Other moieties to whichantibodies may be conjugated include radionuclides such as ³H, ¹²⁵I,¹³¹I ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ³⁶Cl, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, and⁹⁹mTc, are other useful labels which can be conjugated to antibodies.Radio-labeled monoclonal antibodies of the present invention areproduced according to well-known methods in the art. For instance,monoclonal antibodies can be iodinated by contact with sodium orpotassium iodide and a chemical oxidizing agent such as sodiumhypochlorite, or an enzymatic oxidizing agent, such as lactoperoxidase.Monoclonal antibodies according to the invention may be labeled withtechnetium-⁹⁹m by ligand exchange process, for example, by reducingpertechnate with stannous solution, chelating the reduced technetiumonto a Sephadex column and applying the antibody to this column or bydirect labeling techniques, e.g., by incubating pertechnate, a reducingagent such as SNCl₂, a buffer solution such as sodium-potassiumphthalate solution, and the antibody.

5.0 Use of Peptides and Monoclonal Antibodies in Immunoassays

It is proposed that the monoclonal antibodies of the present inventionwill find useful application in standard immunochemical procedures, suchas ELISA and western blot methods, as well as other procedures which mayutilize antibodies specific to CopB epitopes. While ELISAs arepreferred, it will be readily appreciated that such assays include RIAsand other non-enzyme linked antibody binding assays or procedures.Additionally, it is proposed that monoclonal antibodies specific to theparticular UspA epitope may be utilized in other useful applications.For example, their use in immunoabsorbent protocols may be useful inpurifying native or recombinant UspA proteins or variants thereof.

It also is proposed that the disclosed UspA1 and UspA2 peptides of theinvention will find use as antigens for raising antibodies and inimmunoassays for the detection of anti-UspA antigen-reactive antibodies.In a variation on this embodiment, UspA1 and UspA2 mutant peptides maybe screened, in immunoassay format, for reactivity against UspA1- orUspA2-specific antibodies, such as MAb 17C7. In this way, a mutationalanalysis of various epitopes may be performed. Results from suchanalyses may then be used to determine which additional UspA1 or UspA2epitopes may be recognized by antibodies and useful in the preparationof potential vaccines for Moraxella.

Diagnostic immunoassays include direct culturing of bodily fluids,either in liquid culture or on a solid support such as nutrient agar. Atypical assay involves collecting a sample of bodily fluid from apatient and placing the sample in conditions optimum for growth of thepathogen. The determination can then be made as to whether the microbeexists in the sample. Further analysis can be carried out to determinethe hemolyzing properties of the microbe.

Immunoassays encompassed by the present invention include, but are notlimited to those described in U.S. Pat. No. 4,367,110 (double monoclonalantibody sandwich assay) and U.S. Pat. No. 4,452,901 (western blot).Other assays include immunoprecipitation of labeled ligands andimmunocytochemistry, both in-vitro and in vivo.

Immunoassays, in their most simple and direct sense, are binding assays.Certain preferred immunoassays are the various types of enzyme linkedimmunosorbent assays (ELISAs) and radioimmunoassays (RIAs) known in theart. Immunohistochemical detection using tissue sections is alsoparticularly useful. However, it will be readily appreciated thatdetection is not limited to such techniques, and western blotting, dotblotting, FACS analyses, and the like may also be used.

In one exemplary ELISA, the anti-UspA antibodies of the invention areimmobilized onto a selected surface exhibiting protein affinity, such asa well in a polystyrene microtiter plate. Then, a test compositionsuspected of containing the desired antigen, such as a clinical sample,is added to the wells. After binding and washing to removenon-specifically bound immune complexes, the bound antigen may bedetected. Detection is generally achieved by the addition of anotherantibody, specific for the desired antigen, that is linked to adetectable label. This type of ELISA is a simple “sandwich ELISA”.Detection may also be achieved by the addition of a second antibodyspecific for the desired antigen, followed by the addition of a thirdantibody that has binding affinity for the second antibody, with thethird antibody being linked to a detectable label.

In another exemplary ELISA, the samples suspected of containing the UspAantigen are immobilized onto the well surface and then contacted withthe anti-UspA antibodies. After binding and appropriate washing, thebound immune complexes are detected. Where the initial antigen specificantibodies are linked to a detectable label, the immune complexes may bedetected directly. Again, the immune complexes may be detected using asecond antibody that has binding affinity for the first antigen specificantibody, with the second antibody being linked to a detectable label.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the primary antibody is used to form secondaryimmune complexes, as described above. After washing, the secondaryimmune complexes are contacted with a third binding ligand or antibodythat has binding affinity for the second antibody, again underconditions effective and for a period of time sufficient to allow theformation of immune complexes (tertiary immune complexes). The thirdligand or antibody is linked to a detectable label, allowing detectionof the tertiary immune complexes thus formed. This system may providefor signal amplification if desired.

Competition ELISAs are also possible in which test samples compete forbinding with known amounts of labeled antigens or antibodies. The amountof reactive species in the unknown sample is determined by mixing thesample with the known labeled species before or during incubation withcoated wells. (Antigen or antibodies may also be linked to a solidsupport, such as in the form of beads, dipstick, membrane or columnmatrix, and the sample to be analyzed applied to the immobilized antigenor antibody.) The presence of reactive species in the sample acts toreduce the amount of labeled species available for binding to the welland thus reduces the ultimate signal.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating or binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes. These are described below.

In coating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period. The wells of theplate will then be washed to remove incompletely adsorbed material. Anyremaining available surfaces of the wells are then “coated” with anonspecific protein that is antigenically neutral with regard to thetest antisera. These include bovine serum albumin (BSA), casein andsolutions of milk powder. The coating allows for blocking of nonspecificadsorption sites on the immobilizing surface and thus reduces thebackground caused by nonspecific binding of antisera onto the surface.

After binding of antigenic material to the well, coating with anon-reactive material to reduce background, and washing to removeunbound material, the immobilizing surface is contacted with theantisera or clinical or biological extract to be tested in a mannerconducive to immune complex (antigen/antibody) formation. Suchconditions preferably include diluting the antisera with diluents suchas BSA, bovine gamma globulin (BGG) and phosphate buffered saline(PBS)/Tween. These added agents also tend to assist in the reduction ofnonspecific background. The layered antisera is then allowed to incubatefor from 2 to 4 hours, at temperatures preferably on the order of 25° to27° C. Following incubation, the antisera-contacted surface is washed soas to remove non-immunocomplexed material. A preferred washing procedureincludes washing with a solution such as PBS/Tween, or borate buffer.

Following formation of specific immunocomplexes between the test samplearid the bound antigen, and subsequent washing, the occurrence and evenamount of immunocomplex formation may be determined by subjecting sameto a second antibody having specificity for the first. Of course, inthat the test sample will typically be of human origin, the secondantibody will preferably be an antibody having specificity in generalfor human IgG. To provide a detecting means, the second antibody willpreferably have an associated enzyme that will generate a colordevelopment upon incubating with an appropriate chromogenic substrate.Thus, for example, one will desire to contact and incubate theantisera-bound surface with a urease or peroxidase-conjugated anti-humanIgG for a period of time and under conditions which favor thedevelopment of immunocomplex formation (e.g., incubation for 2 hours atroom temperature in a PBS-containing solution such as PBS-Tween).

After incubation with the second enzyme-tagged antibody, and subsequentto washing to remove unbound material, the amount of label is quantifiedby incubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azino-di-(3-ethyl-benzthiazolirne-6-sulfonic acid [ABTS]and H₂O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generation, e.g.,using a visible spectra spectrophotometer. Alternatively, the label maybe a chemilluminescent one. The use of such labels is described in U.S.Pat. Nos. 5,310,687, 5,238,808 and 5,221,605.

6.0 Prophylactic Use of UspA Peptides and UspA-Specific Antibodies

In a further embodiment of the present invention, there are providedmethods for active and passive immunoprophylaxis. Activeimmunoprophylaxis will be discussed first, followed by a discussion onpassive immunoprophylaxis. It should be noted that the discussion offormulating vaccine compositions in the context of active immunotherapyis relevant to the raising antibodies in experimental animals forpassive immunotherapy and for the generation of diagnostic methods.

6.1 Active Immunotherapy

According to the present invention, UspA1 or UspA2 polypeptides orUspA1- or UspA2-derived peptides, as discussed above, may be used asvaccine formulations to generate protective anti-M. catarrhalis antibodyresponses in vivo. By protective, it is only meant that the immunesystem of a treated individual is capable of generating a response thatreduces, to any extent, the clinical impact of the bacterial infection.This may range from a minimal decrease in bacterial burden to outrightprevention of infection. Ideally, the treated subject will not exhibitthe more serious clinical manifestations of M. catarrhalis infection.

Generally, immunoprophylaxis involves the administration, to a subjectat risk, of a vaccine composition. In the instant case, the vaccinecomposition will contain a UspA1 and/or UspA2 polypeptide or immunogenicderivative thereof in a pharmaceutically acceptable carrier, diluent orexcipient. As stated above, those of skill in the art are able, througha variety of mechanisms, to identify appropriate antigeniccharacteristics of UspA1 and UspA2 and, in so UspA2 polypeptide orimmunogenic derivative thereof in a pharmaceutically acceptable carrier,catarrhalis.

The stability and immunogenicity of UspA1 and UspA2 antigens may varyand, therefore, it may be desirable to couple the antigen to a carriermolecule. Exemplary carriers are KLH, BSA, human serum albumin,myoglobin, β-galactosidase, penicillinase, CRM₁₉₇ and bacterial toxoids,such as diphtheria toxoid and tetanus toxoid. Those of skill in the artare aware of proper methods by which peptides can be linked to carrierswithout destroying their immunogenic value. Synthetic carriers such asmulti-poly-DL-alanyl-poly-L-lysine and poly-L-lysine also arecontemplated. Coupling generally is accomplished through amino orcarboxyl-terminal residues of the antigen, thereby affording the peptideor polypeptide the greatest chance of assuming a relatively “native”conformation following coupling.

It is recognized that other protective agents could be coupled witheither a UspA1 or UspA2 antigen such that the UspA1 or UspA2 antigenacts as the carrier molecule. For example, agents which protect againstother pathogenic organisms, such as bacteria, viruses or parasites,could be coupled to either a UspA1 or UspA2 antigen to produce amultivialent vaccine or pharmaceutical composition which would be usefulfor the treatment or inhibition of both M. catarrhalis infection andother pathogenic infections. In particular, it is envisioned that eitherUspA1 or UspA2 proteins or peptides could serve as immunogenic carriersfor other vaccine components, for example, saccharides of pneumococcus,menigococcus or hemophylus influenza and could even be covalentlycoupled to these other components.

It also may be desirable to include in the composition any of a numberof different substances referred to as adjuvants, which are known tostimulate the appropriate portion of the immune system of the vaccinatedanimal. Suitable adjuvants for the vaccination of subjects (includingexperimental animals) include, but are not limited to oil emulsions suchas Freund's complete or incomplete adjuvant (not suitable for livestockuse), Marcol 52: Montanide 888 (Marcol is a Trademark of Esso, Montanideis a Trademark of SEPPIC, Paris), squalane or squalene, Adjuvant 65(containing peanut oil, mannide monooleate and aluminum monostearate),MPL™ (3-O-deacylated monophosphoryl lipid A; RIBI ImmunoChem ResearchInc., Hamilton, Utah), Stimulon™ (QS-21; Aquila Biopharmaceuticals Inc.,Wooster, Mass.), mineral gels such as aluminum hydroxide, aluminumphosphate, calcium phosphate and alum, surfactants such ashexadecylamine, octadecylamine, lysolecithin,dimethyldioctadecylammonium bromide,N,N-dioctadecyl-N,N′-bis(2-hydroxyethyl)-propanediamine,methoxyhexadecylglycerol and pluronic polyols, polyanions such as pyran,dextran sulfate, polyacrylic acid and carbopol, peptides and amino acidssuch as muramyl dipeltide, dimethylglycine, tuftsin and trehalosedimycolate. Agents include synthetic polymers of sugars (Carbopol),emulsion in physiologically acceptable oil vehicles such as mannidemono-oleate (Aracel A) or emulsion with 20 percent solution of aperfluorocarbon (Fluosol-DA) also may be employed.

The preparation of vaccines which contain peptide sequences as activeingredients is generally well understood in the art, as exemplified byU.S. Pat. Nos. 4,608,251; 4,601,903; 4,599,231; 4,599,230; 4,596,792;and 4,578,770, all incorporated herein by reference. Typically, suchvaccines are prepared as injectables. Either as liquid solutions orsuspensions: Solid forms suitable for solution in, or suspension in,liquid prior to injection may also be prepared. The preparation may alsobe emulsified. The active immunogenic ingredient is often mixed withexcipients which are pharmaceutically acceptable and compatible with theactive ingredient. Suitable excipients are, for example, water, saline,dextrose, glycerol, ethanol, or the like and combinations thereof. Inaddition, if desired, the vaccine may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents, pH buffering agents,or adjuvants which enhance the effectiveness of the vaccines. the likeand combinations thereof. In addition, if desired, the vaccine maycontain minor incorporation into non-toxic carriers such as liposomes orother microcarrier substances, or after conjugation to polysaccharides,proteins or polymers or in combination with Quil-A to form “iscoms”(immunostimulating complexes). These complexes can serve to reduce thetoxicity of the antigen, delay its clearance from the host and improvethe immune response by acting as an adjuvant. Other suitable adjuvantsfor use this embodiment of the present invention include INF, IL-2,IL-4, IL-8, IL-12 and other immunostimulatory compounds. Further,conjugates comprising the immunogen together with an integral membraneprotein of prokaryotic origin, such as TraT (see PCT/AU87/00107) mayprove advantageous.

The vaccines are conventionally administered parenterally, by injection,for example, either subcutaneously or intramuscularly. Additionalformulations which are suitable for other modes of administrationinclude suppositories and, in some cases, oral formulations. Forsuppositories, traditional binders and carriers may include, forexample, polyalkalene glycols or triglycerides: such suppositories maybe formed from mixtures containing the active ingredient suppositories,traditional binders and carriers may include, for example, polyalkaleneglycols, or employed excipients as, for example, pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate and the like. These compositions take theform of solutions, suspensions, tablets, pills, capsules, sustainedrelease formulations or powders and contain 10-95% of active ingredient,preferably 25-70%.

The peptides may be formulated into the vaccine as neutral or saltforms.

Pharmaceutically acceptable salts, include the acid addition salts(formed with the free amino groups of the peptide) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic, oxalic, tartaric, mandelic, andthe like. Salts formed with the free carboxyl groups may also be derivedfrom inorganic bases such as, for example, sodium, potassium, ammonium,calcium, or ferric hydroxides, and such organic bases as isopropylamine,trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like.

The vaccines are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective andimmunogenic. The quantity to be administered depends on the subject tobe treated, including, e.g., the capacity of the individual's immunesystem to synthesize antibodies, and the degree of protection desired.Precise amounts of active ingredient required to be administered dependon the judgment of the practitioner. However, suitable dosage ranges areof the order of several hundred micrograms active ingredient pervaccination. Suitable regimes for initial administration and boostershots are also variable, but are typified by an initial administrationfollowed by subsequent inoculations or other administrations.

The manner of application may be varied widely. Any of the conventionalmethods for administration of a vaccine are applicable. These arebelieved to include oral application on a solid physiologicallyacceptable base or in a physiologically acceptable dispersion,parenterally, by injection or the like. The dosage of the vaccine willdepend on the route of administration and will vary according to thesize of the host.

In many instances, it will be desirable to have multiple administrationsof the vaccine, usually not exceeding six vaccinations, more usually notexceeding four vaccinations and preferably one or more, usually at leastabout three vaccinations. The vaccinations will normally be at from twoto twelve week intervals, more usually from three to five weekintervals. Periodic boosters at intervals of 1-5 years, usually threeyears, will be desirable to maintain protective levels of theantibodies. The course of the immunization may be followed by assays forantibodies for the supernatant antigens. The assays may be performed bylabeling with conventional labels, such as radionuclides, enzymes,fluorescers, and the like. These techniques are well known and may befound in a wide variety of patents, such as U.S. Pat. Nos. 3,791,932;4,174,384 and 3,949,064, as illustrative of these types of assays.

6.2 Passive Immunotherapy

Passive immunity is defined, for the purposes of this application, asthe transfer to an organism of an immune response effector that wasgenerated in another organism. The classic example of establishingpassive immunity is to transfer antibodies produced in one organism intoa second, immunologically compatible animal. By “immunologicallycompatible,” it is meant that the antibody can perform at least some ofits immune functions in the new host animal. More recently, as a betterunderstanding of cellular immune functions has evolved, it has becomepossible to accomplish passive immunity by transferring other effectors,such as certain kinds of lymphocytes, including cytotoxic and helper Tcells, NK cells and other immune effector cells. The present inventioncontemplates both of these approaches.

Antibodies, antisera and immune effector cells are raised using standardvaccination regimes in appropriate animals, as discussed above. Theprimary animal is vaccinated with at least a microbe preparation or onebacterial product or by-product according to the present invention, withor without an adjuvant, to generate an immune response. The immuneresponse may be monitored, for example, by measurement of the levels ofantibodies produced, using standard ELISA methods.

Once an adequate immune response has been generated, immune effectorcells can be collected on a regular basis, usually from blood draws. Theantibody fraction can be purified from the blood by standard means,e.g., by protein A or protein G chromatography. In an alternativepreferred embodiment, monoclonal antibody-producing hybridomas areprepared by standard means (Coligan et al., 1991). Monoclonal antibodiesare then prepared from the hybridoma cells by standard means. If theprimary hosts monoclonal antibodies are not compatible with the animalto be treated, it is possible that genetic engineering of the cells canbe employed to modify the antibody to be tolerated by the animal to betreated. In the human context, murine antibodies, for example, may be“humanized” in this fashion.

Antibodies, antisera or immune effector cells, prepared as set forthabove, are injected into hosts to provide passive immunity againstmicrobial infestation. For example, an antibody composition is preparedby mixing, preferably homogeneously mixing, at least one antibody withat least one pharmaceutically or veterinarally acceptable carrier,diluent, or excipient using standard methods of pharmaceutical orveterinary preparation. The amount of antibody required to produce asingle dosage form will vary depending upon the microbial species beingvaccinated against, the individual to be treated and the particular modeof administration. The specific dose level for any particular individualwill depend upon a variety of factors including the age, body weight,general health, sex, and diet of the individual, time of administration,route of administration, rate of excretion, drug combination and theseverity of the microbial infestation.

The antibody composition may be administered intravenously,subcutaneously, intranasally, orally, intramuscularly, vaginally,rectally, topically or via any other desired route. Repeated dosings maybe necessary and will vary, for example, depending on the clinicalsetting, the particular microbe, the condition of the patient and theuse of other therapies.

6.3 DNA Immunization HC

The invention also relates to a vaccine comprising a nucleic acidmolecule encoding a UspA1, UspA2 protein or a peptide comprising SEQ IDNO:17 wherein said UspA1, UspA2 protein or peptide retainsimmunogenicity and, when incorporated into an immunogenic composition orvaccine and administered to a vertebrate, provides protection withoutinducing enhanced disease upon subsequent infection of the vertebratewith M. catarrhalis, and a physiologically acceptable vehicle. Such avaccine is referred to herein as a nucleic acid vaccine or DNA vaccineand is useful for the genetic immunization of vertebrates.

The term, “genetic immunization”, as used herein, refers to inoculationof a vertebrate, particularly a mammal such as a mouse or human, with anucleic acid vaccine directed against a pathogenic agent, particularlyM. catarrhalis, resulting in protection of the vertebrate against M.catarrhalis. A “nucleic acid vaccine” or “DNA vaccine” as used herein,is a nucleic acid construct comprising a nucleic acid molecule encodingUspA1, UspA2 or an immunogenic epitope comprising SEQ ID NO:17. Thenucleic acid construct can also include transcriptional promoterelements, enhancer elements, splicing signals, termination andpolyadenylation signals, and other nucleic acid sequences.

The nucleic acid vaccine can be produced by standard methods. Forexample, using known methods, a nucleic acid (e.g., DNA) encoding UspA1or UspA2 can be inserted into an expression vector to construct anucleic acid vaccine (see Maniatis et al., 1989). The individualvertebrate is inoculated with the nucleic acid vaccine (i.e., thenucleic acid vaccine is administered), using standard methods. Thevertebrate can be inoculated subcutaneously, intravenously,intraperitoneally, intradermally, intramuscularly, topically, orally,rectally, nasally, buccally, vaginally, by inhalation spray, or via animplanted reservoir in dosage formulations containing conventionalnon-toxic, physiologically acceptable carriers or vehicles.Alternatively, the vertebrate is inoculated with the nucleic acidvaccine through the use of a particle acceleration instrument (a “genegun”). The form in which it is administered (e.g., capsule, tablet,solution, emulsion) will depend in part on the route by which it isadministered. Alternatively, the vertebrate is inoculated with thenucleic acid vaccine through the use of a

The nucleic acid vaccine can be administered in conjunction with anysuitable adjuvant. The adjuvant is administered in a sufficient amount,which is that amount that is sufficient to generate an enhanced immuneresponse to the nucleic acid vaccine. The adjuvant can be administeredprior to (e.g., 1 or more days before) inoculation with the nucleic acidvaccine; concurrently with (e.g., within 24 hours of) inoculation withthe nucleic acid vaccine; contemporaneously (simultaneously) with thenucleic acid vaccine (e.g., the adjuvant is mixed with the nucleic acidvaccine, and the mixture is administered to the vertebrate); or after(e.g., 1 or more days after) inoculation with the nucleic acid vaccine.The adjuvant can also be administered at more than one time (e.g., priorto inoculation with the nucleic acid vaccine and also after inoculationwith the nucleic acid vaccine). As used herein, the term “in conjunctionwith” encompasses any time period, including those specificallydescribed herein and combinations of the time periods specificallydescribed herein, during which the adjuvant can be administered so as togenerate an enhanced immune response to the nucleic acid vaccine (e.g.,an increased antibody titer to the antigen encoded by the nucleic acidvaccine, or an increased antibody titer to M. catarrhalis). The adjuvantand the nucleic acid vaccine can be administered at approximately thesame location on the vertebrate; for example, both the adjuvant and thenucleic acid vaccine are administered at a marked site on a limb of thevertebrate.

In a particular embodiment, the nucleic acid construct isco-administered with a transfection-facilitating agent. In a preferredembodiment, the transfection-facilitating agent is dioctylglycylspermine(DOGS) (as exemplified in published PCT application publication no. WO96/21356 and incorporated herein by reference). In another embodiment,the transfection-facilitating agent is bupivicaine (as exemplified inU.S. Pat. No. 5,593,972 and incorporated herein by reference).

6.4 Animal Model for Testing Efficacy of Therapies

The evaluation of the functional significance of antibodies to surfaceantigens of M. catarrhalis has been hampered by the lack of a suitableanimal model. The relative lack of virulence of this organism foranimals rendered identification of an appropriate model system difficult(Doern, 1986). Attempts to use rodents, including chinchillas, to studymiddle ear infections caused by M. catarrhalis were unsuccessful, likelybecause this organism cannot grow or survive in the middle ear of thesehosts (Doyle, 1989).

Murine short-term pulmonary clearance models have now been developed(Unhanand et al., 1992; Verghese et al., 1990) which permit anevaluation of the interaction of M. cattrrhalis with the lowerrespiratory tract as well as assessment of pathologic changes in thelungs. This model reproducibly delivers an inoculum of bacteria to alocalized oeripherial segment of the murine lung. Bacteria multiplywithin the lung, but are eventually cleared as a result of (i) residentdefense mechanisms, (ii) the development of an inflammatory response,and/or (iii) the development of a specific immune response. Using thismodel, it has been demonstrated that serum IgG antibody can enter thealveolar spaces in the absence of an inflammatory response and enhancepulmonary clearance of nontypable H. influenzae (McGehee et al., 1989),a pathogen with a host range and disease spectrum nearly identical tothose of M. catarrhalis.

7.0 Screening Assays

In still further embodiments, the present invention provides methods foridentifying new M. catarrhalis inhibitory compounds, which may be termedas “candidate substances,” by screening for immunogenic activity withpeptides that include one or more mutations to the identifiedimmunogenic epitopic region. It is contemplated that such screeningtechniques will prove useful in the general identification of anycompound that will serve the purpose of inhibiting, or even killing, M.catarrhalis, and in preferred embodiments, will provide candidatevaccine compounds.

It is further contemplated that useful compounds in this regard will inno way be limited to proteinaceous or peptidyl compounds. In fact, itmay prove to be the case that the most useful pharmacological compoundsfor identification through application of the screening assays will benon-peptidyl in nature and, e.g., which will serve to inhibit bacterialprotein transcription through a tight binding or other chemicalinteraction. Candidate substances may be obtained from libraries ofsynthetic chemicals, or from natural samples, such as rain forest andmarine samples.

To identify a M. catarrhalis inhibitor, one would simply conductparallel or otherwise comparatively controlled immunoassays and identifya compound that inhibits the phenotype of M. catarrhalis. Those of skillin the art are familiar with the use of immunoassays for competitivescreenings (for example refer to Sambrook et al. 1989).

Once a candidate substance is identified, one would measure the abilityof the candidate substance to inhibit M. catarrhalis in the presence ofthe candidate substance. In general, one will desire to measure orotherwise determine the activity of M. catarrhalis in the absence of theadded candidate substance relative to the activity in the presence ofthe candidate substance in order to assess the relative inhibitorycapability of the candidate substance.

7.1 Mutagenesis

Site-specific mutagenesis is a technique useful in the preparation ofindividual peptides, or biologically functional equivalent proteins orpeptides, through specific mutagenesis of the underlying DNA. Thetechnique further provides a ready ability to prepare and test sequencevariants, incorporating one or more of the foregoing considerations, byintroducing one or more nucleotide sequence changes into the DNA.Site-specific mutagenesis allows the production of mutants through theuse of specific oligonucleotide sequences which encode the DNA sequenceof the desired mutation, as well as a sufficient number of adjacentnucleotides, to provide a primer sequence of sufficient size andsequence complexity to form a stable duplex on both sides of thedeletion junction being traversed. Typically, a primer of about 17 to 25nucleotides in length is preferred, with about 5 to 10 residues on bothsides of the junction of the sequence being altered.

In general, the technique of site-specific mutagenesis is well known inthe art, as will be appreciated, the technique typically employs abacteriophage vector that exists in both a single stranded and doublestranded form. Typical vectors useful in site-directed mutagenesisinclude vectors such as the M13 phage. These phage vectors arecommercially available and their use is generally well known to thoseskilled in the art. Double stranded plasmids are also routinely employedin site directed mutagenesis, which eliminates the step of transferringthe gene of interest from a phage to a plasmid. generally well known tothose skilled in the art. Double stranded plasmids are also routinelyvector, or melting of two strands of a double stranded vector whichincludes within its sequence a DNA sequence encoding the desiredprotein. An oligonucleotide primer bearing the desired mutated sequenceis synthetically prepared. This primer is then annealed with thesingle-stranded DNA preparation, and subjected to DNA polymerizingenzymes such as E. coli polymerase I Klenow fragment, in order tocomplete the synthesis of the mutation-bearing strand. Thus, aheteroduplex is formed wherein one strand encodes the originalnon-mutated sequence and the second strand bears the desired mutation.This heteroduplex vector is then used to transform appropriate cells,such as E. coli cells, and clones are selected that include recombinantvectors bearing the mutated sequence arrangement.

The preparation of sequence variants of the selected gene usingsite-directed mutagenesis is provided as a means of producingpotentially useful species and is not meant to be limiting, as there areother ways in which sequence variants of genes may be obtained. Forexample, recombinant vectors encoding the desired gene may be treatedwith mutagenic agents, such as hydrox ylamine, to obtain sequencevariants.

7.2 Second Generation Inhibitors

In addition to the inhibitory compounds initially identified, theinventor also contemplates that other sterically similar compounds maybe formulated to mimic the key portions of the structure of theinhibitors. Such compounds, which may include peptidomimetics of peptideinhibitors, may be used in the same manner as the initial inhibitors.

Certain mimetics that mimic elements of protein secondary structure aredesigned using the rationale that the peptide backbone of proteinsexists chiefly to orientate amino acid side chains in such a way as tofacilitate molecular interactions. A peptide mimetic is thus designed topermit molecular interactions similar to the natural molecule.

Some successful applications of the peptide mimetic concept have focusedon mimetics of β-turns within proteins, which are known to be highlyantigenic. Likely β-turn structure within a polypeptide can be predictedby computer-based algorithms, as discussed herein. Once the componentamino acids of the turn are determined, mimetics can be constructed toachieve a similar spatial orientation of the essential elements of theamino acid side chains.

The generation of further structural equivalents or mimetics may beachieved by the techniques of modeling and chemical design known tothose of skill in the art. The art of computer-based chemical modelingis now well known. Using such methods, a chemical that specificallyinhibits viral transcription elongation can be designed, and thensynthesized, following the initial identification of a compound thatinhibits RNA elongation, but that is not computer-based chemicalmodeling is now well known. Using such methods, a chemical thatelongation. It will be understood that all such sterically similarconstructs and second generation molecules fall within the scope of thepresent invention.

8.0 Diagnosing M. catarrhalis Infections 8.1 Amplification and PCR™

Nucleic acid sequence used as a template for amplification is isolatedfrom cells contained in the biological sample, according to standardmethodologies (Sambrook et al., 1989). The nucleic acid may be genomicDNA or fractionated or whole cell RNA. Where RNA is used, it may bedesired to convert the RNA to a cDNA.

Pairs of primers that selectively hybridize to nucleic acidscorresponding to UspA1 or UspA2 protein or a mutant thereof arecontacted with the isolated nucleic acid under conditions that permitselective hybridization. The term “primer”, as defined herein, is meantto encompass any nucleic acid that is capable of priming the synthesisof a nascent nucleic acid in a template-dependent process. Typically,primers are oligonucleotides from ten to twenty base pairs in length,but longer sequences can be employed. Primers may be provided indouble-stranded or single-stranded form, although the single-strandedform is preferred.

Once hybridized, the nucleic acid:primer complex is contacted with oneor more enzymes that facilitate template-dependent nucleic acidsynthesis. Multiple rounds of amplification, also referred to as“cycles,” are conducted until a sufficient amount of amplificationproduct is produced.

Next, the amplification product is detected. In certain applications,the detection may be performed by visual means. Alternatively, thedetection may involve indirect identification of the product viachemiluminescence, radioactive scintigraphy of incorporated radiolabelor fluorescent label or even via a system using electrical or thermalimpulse signals (Affymax technology).

A number of template dependent processes are available to amplify themarker sequences present in a given template sample. One of the bestknown amplification methods is the polymerase chain reaction (referredto as PCR™) which is described in detail in U.S. Pat. Nos. 4,683,195,4,683,202 and 4,800,159, and each incorporated herein by reference inentirety. the polymerase chain reaction (referred to as PCR™) which isdescribed in detail in U.S. Pat. on opposite complementary strands ofthe marker sequence. An excess of deoxynucleoside triphosphates areadded to a reaction mixture along with a DNA polymerase, e.g., Taqpolymerase. If the marker sequence is present in a sample, the primerswill bind to the marker and the polymerase will cause the primers to beextended along the marker sequence by adding on nucleotides. By raisingand lowering the temperature of the reaction mixture, the extendedprimers will dissociate from the marker to form reaction products,excess primers will bind to the marker and to the reaction products andthe process is repeated.

A reverse transcriptase PCR™ (RT-PCR™) amplification procedure may beperformed in order to quantify the amount of mRNA amplified or toprepare cDNA from the desired mRNA. Methods of reverse transcribing RNAinto cDNA are well known and described in Sambrook et al., 1989.Alternative methods for reverse transcription utilize thermoostable, inorder to quantify the amount of RNA amplified or to prepare cDNA fromthe desired RNA. Methods of reverse transcribing RNA into cDNA are wellknown and described in methodologies are well known in the art.

Another method for amplification is the ligase chain reaction (“LCR”),disclosed in EPA No. 320 308, incorporated herein by reference in itsentirety. In LCR, two complementary probe pairs are prepared, and in thepresence of the target sequence, each pair will bind to oppositecomplementary strands of the target such that they abut. In the presenceof a ligase, the two probe pairs will link to form a single unit. Bytemperature cycling, as in PCR™, bound ligated units dissociate from thetarget and then serve as “target sequences” for ligation of excess probepairs. U.S. Pat. No. 4,883,750 describes a method similar to LCR forbinding probe pairs to a target sequence.

Qbeta Replicase, described in PCT Application No. PCT/US87/00880,incorporated herein by reference, may also be used as still anotheramplification method in the present invention. In this method, areplicative sequence of RNA that has a region complementary to that of atarget is added to a sample in the presence of an RNA polymerase. Thepolyinerase will copy the replicative sequence that can then bedetected.

An isothermal amplification method, in which restriction endonucleasesand ligases are used to achieve the amplification of target moleculesthat contain nucleotide 5′-[alpha-thio]-triphosphates in one strand of arestriction site may also be useful in the amplification of nucleicacids in the present invention.

Strand Displacement Amplification (SDA) is another method of carryingout isothermal amplification of nucleic acids which involves multiplerounds of strand displacement and synthesis, i.e., nick translation. Asimilar method, called Repair Chain Reaction (PCR), involves annealingseveral probes throughout a region targeted for amplification, followedby a repair reaction in which only two of the four bases are present.The other two bases can be synthesis, i.e., nick translation. A similarmethod, called Repair Chain Reaction (PCR), specific sequences can alsobe detected using a cyclic probe reaction (CPR). In CPR, a probe having3′ and 5′ sequences of non-specific DNA and a middle sequence ofspecific RNA is hybridized to DNA that is present in a sample. Uponhybridization, the reaction is treated with RNase H, and the products ofthe probe identified as distinctive products that are released afterdigestion. The original template is annealed to another cycling probeand the reaction is repeated.

Still another amplification methods described in GB Application No. 2202 328, and in PCT Application No. PCT/US89/01025, each of which isincorporated herein by reference in its entirety, may be used inaccordance with the present invention. In the former application,“modified” primers are used in a PCR™-like, template- andenzyme-dependent synthesis. The primers may be modified by labeling witha capture moiety (e.g., biotin) and/or a detector moiety (e.g., enzyme).In the latter application, an excess of labeled probes are added to asample. In the presence of the target sequence, the probe binds and iscleaved catalytically. After cleavage, the target sequence is releasedintact to be bound by excess probe. Cleavage of the labeled probesignals the presence of the target sequence.

Other nucleic acid amplification procedures include transcription-basedamplification systems (TAS), including nucleic acid sequence basedamplification (NASBA) and 3SR Gingeras et al., PCT Application WO88/10315, incorporated herein by reference. In NASBA, the nucleic acidscan be prepared for amplification by standard phenol/chloroformextraction, heat denaturation of a clinical sample, treatment with lysisbuffer and minispin columns for isolation of DNA and RNA or guanidiniumchloride extraction of RNA. These amplification techniques involveannealing a primer which has target specific sequences. Followingpolymerization, DNA/RNA hybrids are digested with RNase H while doublestranded DNA molecules are heat denatured again. In either case thesingle stranded DNA is made fully double stranded by addition of secondtarget specific primer, followed by polymerization. The double-strandedDNA molecules are then multiply transcribed by an RNA polymerase such asT7 or SP6. In an isothermal cyclic reaction, the RNA's are reversetranscribed into single stranded DNA, which is then converted to doublestranded DNA, and then transcribed once again with an RNA polymerasesuch as T7 or SP6. The resulting products, whether truncated orcomplete, indicate target specific sequences.

Davey et al., EPA No. 329 822 (incorporated herein by reference in itsentirety) disclose a nucleic acid amplification process involvingcyclically synthesizing single-stranded RNA (“ssRNA”), ssDNA, anddouble-stranded DNA (dsDNA), which may be used in accordance with thepresent invention. The ssRNA is a template for a first primeroligonucleotide, which is elongated by reverse transcriptase(RNA-dependent DNA polymerase). The RNA is then removed from theresulting DNA:RNA duplex by the action of ribonuclease H (RNase H, anRNase specific for RNA in duplex with either DNA or RNA). The resultantssDNA is a template for a second primer, which also includes thesequences of an RNA polyruerase promoter (exemplified by T7 RNApolymerase) 5′ to its homology to the template. This primer is thenextended by DNA polymerase (exemplified by the large “Klenow” fragmentof E. coli DNA polymerase I), resulting in a double-stranded DNA(“dsDNA”) molecule, halving a sequence identical to that of the originalRNA between the primers and having additionally, at one end, a promotersequence. This promoter sequence can be used by the appropriate RNApolymerase to make many RNA copies of the DNA. These copies can thenre-enter the cycle leading to very swift amplification. With properchoice of enzymes, this amplification can be done isothermally withoutaddition of enzymes at each cycle. Because of the cyclical nature ofthis process, the starting sequence can be chosen to be in the form ofeither DNA or RNA.

Miller et al., PCT Application WO 89/06700 (incorporated herein byreference in its entirety) disclose a nucleic acid sequenceamplification scheme based on the hybridization of a promoter/primersequence to a target single-stranded DNA (“ssDNA”) followed bytranscription of many RNA copies of the sequence. This scheme is notcyclic, i.e., new templates are not produced from the resultant RNAtranscripts. Other amplification methods include “RACE” and “one-sidedPCR” (Frohman, 1990, incorporated by reference).

Methods based on ligation of two (or more) oligonucleotides in thepresence of nucleic acid having the sequence of the resulting“di-oligonucleotide”, thereby amplifying the di-oligonucleotide, mayalso be used in the amplification step of the present invention.

Following any amplification, it may be desirable to separate theamplification product from the template and the excess primer for thepurpose of determining whether specific amplification has occurred. Inone embodiment, amplification products are separated by agarose,agarose-acrylamide or polyacrylamide gel electrophoresis using standardmethods. See Sambrook et al., 1989.

Alternatively, chromatographic techniques may be employed to effectseparation. There are many kinds of chromatography which may be used inthe present invention: adsorption, partition, ion-exchange and molecularsieve, and many specialized techniques for using them including column,paper, thin-layer and gas chromatography.

Amplification products must be visualized in order to confirmamplification of the marker sequences. One typical visualization methodinvolves staining of a gel with ethidium bromide and visualization underUV light. Alternatively, if the amplification products are integrallylabeled with radio- or fluorometrically-labeled nucleotides, theamplification products can then be exposed to x-ray film or visualizedunder the appropriate stimulating spectra, following separation.

In one embodiment, visualization is achieved indirectly. Followingseparation of amplification products, a labeled, nucleic acid probe isbrought into contact with the amplified marker sequence. The probepreferably is conjugated to a chromophore but may be radiolabeled. Inanother embodiment, the probe is conjugated to a binding partner, suchas an antibody or biotin, and the other member of the binding paircarries a detectable moiety.

In one embodiment, detection is by Southern blotting and hybridizationwith a labeled probe. The techniques involved in Southern blotting arewell known to those of skill in the art and can be found in manystandard books on molecular protocols. See Sambrook et al., 1989.Briefly, amplification products are separated by gel electrophoresis.The gel is then contacted with a membrane, such as nitrocellulose,permitting transfer of the nucleic acid and noncovalent binding.Subsequently, the membrane is incubated with a chromophore-conjugatedprobe that is capable of hybridizing with a target amplificationproduct. Detection is by exposure of the membrane to x-ray film orion-emitting detection devices.

One example of the foregoing is described in U.S. Pat. No. 5,279,721,incorporated by reference herein, which discloses an apparatus andmethod for the automated electrophoresis and transfer of nucleic acids.The apparatus permits electrophoresis and blotting without externalmanipulation of the gel and is ideally suited to carrying out methodsaccording to the present invention.

All the essential materials and reagents required for detecting P-TEFbor kinase protein markers in a biological sample may be assembledtogether in a kit. This generally will comprise preselected primers forspecific markers. Also included may be enzymes suitable for amplifingnucleic acids including various polymerases (RT, Taq, etc.),deoxynucleotides and buffers to provide the necessary reaction mixturefor amplification.

Such kits generally will comprise, in suitable means, distinctcontainers for each individual reagent and enzyme as well as for eachmarker primer pair. Preferred pairs of primers for amplifying nucleicacids are selected to amplify the sequences specified in SEQ ID NO:2 orSEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10 or SEQ IDNO:12 or SEQ ID NO:14 or SEQ ID NO:16 such that, for example, nucleicacid fragments are prepared that include a contiguous stretch ofnucleotides identical to for example about 15, 20, 25, 30, 35, etc.; 48,49, 50, 51, etc.; 75, 76, 77, 78, 79, 80 etc.; 100, 101, 102, 103 etc.;118, 119, 120, 121 etc.; 127, 128, 129, 130, 131, etc.; 316, 317, 318,319, etc.; 322, 323, 324, 325, 326, etc.; 361, 362, 363, 364, etc.; 372,373, 374, 375, etc. of SEQ ID NO:2 or SEQ ID NO:4 or SEQ ID NO:6 or SEQID NO:8 or SEQ ID NO:10 or SEQ ID NO:12 or SEQ ID NO:14 or SEQ ID NO:16,so long as the selected contiguous stretches are from spatially distinctregions. Similar ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10 or SEQ ID NO:12or SEQ ID NO:14 or SEQ ID NO:1 such that the fragments do not hybridizeto, for example, SEQ ID NO:3.

In another embodiment, such kits will comprise hybridization probesspecific for UspA1 or UspA2 proteins chosen from a group includingnucleic acids corresponding to the sequences specified in SEQ ID NO:2 orSEQ ID NO:4 or SEQ ID NO:6 or SEQ ID NO:8 or SEQ ID NO:10 or SEQ IDNO:12 or SEQ ID NO:14 or SEQ ID NO:16 or to intermediate lengths of thesequences specified. Such kits generally will comprise, in suitablemeans, distinct containers for each individual reagent and enzyme aswell as for each marker hybridization probe.

8.2 Other Assays

Other methods for genetic screening to accurately detect M. catarrhalisinfections that alter normal cellular production and processing, ingenomic DNA, cDNA or RNA samples may be employed, depending on thespecific situation.

For example, one method of screening for genetic variation is based onRNase cleavage alter normal cellular production and processing, ingenomic DNA, cDNA or RNA samples may term “mismatch” is defined as aregion of one or more unpaired or mispaired nucleotides in adouble-stranded RNA/RNA, RNA/DNA or DNA/DNA molecule. This definitionthus includes mismatches due to insertion/deletion mutations, as well assingle and multiple base point mutations.

U.S. Pat. No. 4,946,773 describes an RNase A mismatch cleavage assaythat involves annealing single-stranded DNA or RNA test samples to anRNA probe, and subsequent treatment of the nucleic acid duplexes withRNase A. After the RNase cleavage reaction, the RNase is inactivated byproteolytic digestion and organic extraction, and the cleavage productsare denatured by heating and analyzed by electrophoresis on denaturingpolyacrylamideo gels. For the detection of mismatches, thesingle-stranded products of the RNase A treatment, electrophoreticallyseparated according to size, are compared to similarly treated controlduplexes. Samples containing smaller fragments (cleavage products) notseen in the control duplex are scored as +.

Currently available RNase mismatch cleavage assays, including thoseperformed according to U.S. Pat. No. 4,946,773, require the use ofradiolabeled RNA probes. Myers and Maniatis in U.S. Pat. No. 4,946,773describe the detection of base pair mismatches using RNase A. Otherinvestigators have described the use of E. coli enzyme, RNase I, inmismatch assays. Because it has broader cleavage specificity than RNaseA, RNase I would be a desirable enzyme to employ in the detection ofbase pair mismatches if components can be found to decrease the extentof non-specific cleavage and increase the frequency of cleavage ofmismatches. The use of RNase I for mismatch detection is described inliterature from Promega Biotech. Promega markets a kit containing RNaseI that is shown in their literature to cleave three out of four knownmismatches, provided the enzyme level is sufficiently high.

The RNase protection assay was first used to detect and map the ends ofspecific mRNA targets in solution. The assay relies on being able toeasily generate high specific activity radiolabeled RNA probescomplementary to the mRNA of interest by in vitro transcription.Originally, the templates for in vitro transcription were recombinantplasmids containing bacteriophage promoters. The probes are mixed withtotal cellular RNA samples to permit radiolabeled RNA probescomplementary to the RNA of interest by in vitro transcription. excessunhybridized probe. Also, as originally intended, the RNase used isspecific for single-stranded RNA, so that hybridized double-strandedprobe is protected from degradation. After inactivation and removal ofthe RNase, the protected probe (which is proportional in amount to theamount of target mRNA that was present) is recovered and analyzed on apolyacrylmide gel.

The RNase Protection assay was adapted for detection of single basemutations. In this type of RNase A mismatch cleavage assay, radiolabeledRNA probes transcribed in vitro from wild type sequences, are hybridizedto complementary target regions derived from test samples. The testtarget generally comprises DNA (either genomic DNA or DNA amplified bycloning in plasmids or by PCR™), although RNA targets (endogenous mRNA)have occasionally been used. If single nucleotide (or greater) sequencedifferences occur between the hybridized probe and target, the resultingdisruption in Watson-Crick hydrogen bonding at that position(“mismatch”) can be recognized and cleaved in some cases bysingle-strand specific ribonuclease. To date, RNase A has been usedalmost exclusively for cleavage of single-base mismatches, althoughRNase I has recently been shown as useful also for mismatch cleavage.There are recent descriptions of using the MutS protein and otherDNA-repair enzymes for detection of single-base mismatches.

9.0 Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example I Sequence Analysis and Characterization of uspA1

Bacterial strains and culture conditions. M. catarrhalis strains O35E,O46E, TTA24, 012E, FR2682, and B21 have been previously described(Helminen et al., 1993a; Helminen et al., 1994; Unhanand et al., 1992).M. catarrhalis strains FR3227 and FR2336 were obtained from RichardWallace, University of Texas Health Center, Tyler, Tex. M. catarrhalisstrain B6 was obtained from Elliot Juni, University of Michigan, AnnArbor, Mich. M. catarrhalis strain TTA1 was obtained from Steven Berk,East Tennessee State University, Johnson City, Tenn. M. catarrhalisstrain 25240 was obtained from the American Type Culture Collection,Rockville, Md. M. catarrhalis strains were routinely cultured in BrainHeart Infusion (BHI) broth (Difco Laboratories, Detroit, Mich.) at 37°C. or on BHI agar plates in an atmosphere of 95% air-5% CO₂ .Escherichia coli strains LE392 and XL1-Blue MRF′ (Stratagene, La Jolla,Calif.) were grown on Lubria-Bertani medium (Maniatis et al., 1982)supplemented with maltose (0.2% w/v) and 10 mM MgSO₄ at 37° C., withantimicrobial supplementation as necessary.

Monoclonal antibodies (MAbs). MAb 17C7 is a murine IgG antibody reactivewith the UspA proteinaceous material of all M. catarrhalis strainstested to date (Helminen et al., 1994). Additional MAbs specific forUspA material (i.e., 16A7, 17B1, and 5C12) were produced for this studyby fusing spleen cells from mice immunized with outer membrane vesiclesfrom M. catarrhalis 035E with the SP2/0-Ag14 plasmacytoma cell line, asdescribed (Helminen et al., 1993a). These MAbs were used in the form ofhybridoma culture supernatant fluid in western blot and dot blotanalyses.

Cloning vectors. Plasmid and bacteriophage cloning vectots utilized inthis work and the recombinant derivatives of these vectors are listed inTable VI.

TABLE VI Bacteriophages and Plasmids Bacteriophage or plasmidDescription Source Bacteriophage LambdaGEM-11 Cloning vector PromegaCorp. (Madison, WI) MEH200 LambdaGEM-11 containing an (Helminen et al.,11 kb insert of M. catarrhalis 1994) strain 035E DNA encoding the UspAproteinaceous material ZAP Express Cloning vector Stratagene USP100 ZAPExpress with a 2.7 kb This study fragment of DNA (containing the uspA1)amplified from the chromosome of M. catarrhalis strain 035E PlasmidspBluescript II SK+ Cloning vector, AMP^(R) Stratagene (pBS) pJL501.6 pBScontaining the 1.6 kb This study BglII-EcoRI fragment from MEH200pJL500.5 pBS containing the 600-bp BglII This study fragment form MEH200

MEH200, the original recombinant bacteriophage clone that producedplaques reactive with the UspA-specific MAb 17C7, has been describedpreviously (Helminen et al., 1994).

Genetic techniques. Standard recombinant DNA techniques includingplasmid isolation, restriction enzyme digestions, DNA modifications,ligation reactions and transformation of E. coli are familiar to thoseof skill in the art and were performed as previously described (Maniatiset al., 1982; Sambrook et al., 1989).

Polymerase Chain Reaction PCR™). PCR™ was performed using the GeneAmpkit (Perkin-Elmer, Branchberg, N.J.). All reaction were carried outaccording to the manufacturer's instructions. To amplify products fromtotal genomic DNA, 1 μg of M. catarrhalis chromosomal DNA and 100 ng ofeach primer were used in each 100 μl reaction.

Nucleotide sequence analysis. Nucleotide sequence analysis of DNAfragments in instructions. To amplify products from total genomic DNA, 1μg of M. catarrhalis Biosystems Model 373A automated DNA sequencer(Applied Biosystems, Foster City, Calif.). DNA sequence information wasanalyzed using the Intelligenetics suite package and programs from theUniversity of Wisconsin Genetics Computer Group software analysispackage (Devereux et al., 1984). Analysis of protein hydrophilicityusing the method of Kyte and Doolittle (1982) and analysis of repeatedamino acid sequences within the UspA protein was performed using theMacVector™ software protein matrix analysis package (Eastman KodakCompany, Rochester, N.Y.).

Identification of recombinant bacteriophage. Lysates were generated fromE. coli cells performed using the MacVector™ software protein matrixanalysis package (Eastman Kodak (Helminen et al., 1994). MAb-basedscreening of plaques formed by recombinant ZAP Express bacteriophage onE. coli XL1-Blue MRF′ cells was performed according to themanufacturer's instructions (Stratagene, La Jolla, Calif.). Briefly,nitrocellulose filters soaked in 10 mM IPTG were applied to the surfaceof agar plates five hours after bacteriophage infection of thebacteriophage on E. coli XL1-Blue MRF′ cells was performed according tothe manufacturer's washed with PBS containing 0.5% (v/v) Tween 20 and 5%(w/v) skim milk (PBS-T) and incubated with hybridoma culture supernatantcontaining the MAb for 4 hours at room temperature. After four washeswith PBS-T, PBS-T containing ¹²⁵I-labeled goat aniti-mouse IgG wasapplied to each pad. After overnight incubation at 4° C., the pads werewashed four times with PBS-T, blotted dry, and exposed to film.

Characterization of M. catarrhalis protein antigens. Outer membranevesicles were prepared from BHI broth-grown M. catarrhalis cells by theEDTA-buffer method (Murphy and Loeb, 1989). Proteins present in thesevesicles were resolved by sodium dodecyl sulfate (SDS)-polyacrylamidegel electrophoresis (PAGE) using 7.5% (w/v) polyacrylamide separatinggels. These SDS-PAGE-resolved proteins were electrophoreticallytransferred to nitrocellulose Loeb, 1989). Proteins present in thesevesicles were resolved by sodium dodecyl sulfate (Kimura et al., 1985).For western blot analysis of proteins encoded by DNA inserts inrecombinant bacteriophage, one part of a lysate frombacteriophage-infected E. coli cells was mixed with one partSDS-digestion buffer (Kimura et al., 1985) and this mixture wasincubated at 37° C. for 15 minutes prior to SDS-PAGE.

Features of the uspA1 gene and its encoded protein product. Thenucleotide sequence of the M. catarrhalis 035E uspA1 gene and thededuced amino acid sequence of the UspA1 protein are provided in SEQ IDNO:2 and SEQ ID NO:1, respectively. The open reading frame (ORF),containing 2,493 nucleotides, encoded a protein product of 831 aminoacids, with a calculated molecular mass of 88,271 daltons.

The predicted protein product of the uspA1 ORF had a pI or 4.7, washighly hydrophilic, and was characterized by oxtcnmiyely repeatedmotifs. The first motif consists of the consensus sequenceNXAXXYSXIGGGXN (SEQ ID NO:24), which is extensively repeated betweenamino acid residues 80 and 170. The second region, from amino acidresidues 320 to 460, contains a long sequence which is repeated threetimes in its entirety, but which also contains smaller units which arerepeated several times themselves. This “repeat within a repeat”arrangement is also true of the third region, which extends from aminoacid residues 460 to 600. This last motif consists of many repeats ofthe small motif QADI (SEQ ID NO:25) and two large repeats which containthe QADI (SEQ ID NO:25) motif within themselves.

Similarity of UspA1 to other proteins. A BLAST-X search (Altschul etal., 1990; Gish and States, 1993) of the available databases forproteins with significant homology to UspA1 indicated that theprokaryotic proteins that were most similar to this M. catarrhalisantigen were a putative adhesion of H. influenzae Rd (GenBank accessionnumber U32792) (Fleischmann et al., 1995), the Hia adhesion fromnontypable H. influenzae (GenBank accession number U38617) (Barenkampand St. Geme III, 1996), and the YadA invasin of Yersinia enterocolitica(Skurnik and Wolf-Watz, 1989) (SwissProt:P31489). When the GAP alignmentprogram (Devereux et al., 1984) was used to compare the UspA1 sequenceto that of these and closely related bacterial adhesins, UspA1 proved tobe 25% identical and 47% similar to the E. coli AIDA-I adhesion fromenteropathogenic E. coli (Benz and Schmidt, 1989; Benz and Schmidt,1992b), 23% identical and 46% similar to Hia (Barenkamp and St. GemeIII, 1996), and 24% identical and 43% similar to YadA (Skurnik andWolf-Watz, 1989). Other proteins retrieved from database searches ashaving homology with UspA1 included myosin heavy chains from a number ofspecies.

Example II Two Genes Encode the Proteins UspA1 and UspA2

MAb 17C7 binds to a very high molecular weight proteinaceous material ofM. catarrhalis, designated UspA, that migrates with an apparentmolecular weight (in SDS-PAGE) of at least 250 kDa. This same MAb alsoreacts with another antigen band of approximately 100 kDa, as describedin U.S. Pat. No. 5,552,146 and incorporated herein by reference, and itis bound by a phage lysate from E. coli infected by a recombinantbacteriophage that contained a fragment of M. catarrhalis chromosomalDNA. The M. catarrhalis proteinaceous material in the phage lysate thatbinds this MAb migrates at a rate similar or indistinguishable from thatof the native UspA material (Helminen et al., 1994).

Analysis of uspA1. Nucleotide sequence analysis of the M. catarrhalisstrain O35E gene expressed by the recombinant bacteriophage, designateduspA1, revealed the presence of an ORF encoding a predicted proteinproduct with a molecular mass of 88,271 (SEQ ID NO:1). The use of theuspA1 ORF in an in vitro DNA-directed protein expression system revealedthat the protein encoded by the uspa1 gene migrated in SDS-PAGE with anapparent molecular weight of about 120 kDa. (Those of skill in the artwill be aware that denaturing processes, such as SDS-PAGE, can alter themigration rate of proteins such that the apparent molecular weight ofthe denatured protein is somewhat different than the predicted molecularweight of the non-denatured protein.) In addition, when the uspA1 ORFwas introduced into a bacteriophage vector, the recombinant E. colistrain containing this recombinant phage expressed a protein thatmigrated in SDS-PAGE apparently at the same rate as the native UspAprotein from M. catarrhalis.

Southern blot analysis of chromosomal DNA from several M. catarrhalisstrains, using a 0.6 kb BglII-PvuII fragment derived from the cloneduspA1 gene as the probe, revealed that, with several strains, there weretwo distinct restriction fragments that bound this uspA1-derived probe(FIG. 1), indicating that M. catarrhalis possessed a second gene hadsome similarity to the uspA1 gene.

Native very high molecular weight UspA proteinaceous material from M.catarrhalis strain O35E was resolved by SDS PAGE, electroeluted,anddigested with a protease. N-terminal acid sequence analysis of some ofthe resultant peptides revealed that the amino acid sequences of severalpeptides did not match that of the deduced amino acid sequence of UspA1.Other peptides obtained from this experiment were similar to thosepresent in the deduced amino acid sequence but not identical.

Protease and cyanogen bromide (CNBr) Cleavage of High Molecular WeightUspA Proteinaceous Material: Three tenths (0.3) mg of purified very highmolecular weight UspA proteinaceous material (at the time of thepurification this material was thought to be a single protein) wasprecipitated with 90% ethanol and the pellet was resuspended in 100 mlof 88% formic acid containing 12M urea. Following resuspension, 100 mlof 88% formic acid containing 2M CNBr was added and the mixture wasincubated in the dark overnight at room temperature. One ml (2.0 mg) ofpurified UspA material was added directly to a vial containing 25 mg ofeither trypsin or chymotrypsin. The reaction mixtures were incubated for˜48 hours. at 37° C. One ml (2.0 mg) of purified UspA material was addeddirectly to a vial containing 15 mg of endoproteinase Lys-C. Thereaction mixtures were incubated for about 48 hours at 37° C.

The cleavage reaction mixtures were clarified by centrifugation in anEppendorf™ centrifuge at 12,000 rpm for 5 minutes. The clarifiedsupernatant was loaded directly onto a Vydac C4 HPLC column using amobile phase of 0.1% (v/v) aqueous trifluoroacetic acid (Solvent A) andacetonitrile:H₂O:trifluoroacetic acid, 80:20:0.1 (v/v/v) (Solvent B) ata flow rate of 1.0 ml/min. The reaction mixtures were washed onto thecolumn with 100% Solvent A followed by elution of cleavage fragmentsusing a 30 minutes linear gradient (0-100%) of Solvent B. Fractions werecollected manually, dried overnight in a Speed-Vac and resuspended inHouse Pure Water. The resuspended HPLC-separated fractions weresubjected to SDS-PAGE analysis using 10-18% gradient gels in aTris-Tricine buffer system. The fractions which exhibited a singlepeptide band were submitted for direct N-terminal sequence analysis.Fractions displaying multiple peptide bands were transferred fromSDS-PAGE onto a, PVDF membrane and individual bands excised andsubmitted for N-terminal sequence analysis.

The N-terminal amino acid sequences of these fragments then weredetermined using an Applied Biosystems Model 477A PTH Analyzer (AppliedBiosystems, Foster City, Calif., U.S.A.). A summary of these sequencesis given in Table VII. About half of the sequences were found to matchthe sequence deduced from the uspA1 gene, while the other half did not.Attempts at shifting the reading frame of the uspa1 gene sequence failedto account for the non-matching peptide sequences, indicating that thehigh molecular weight UspA protein may comprise either a multimer ofmore than one distinct protein or distinct multimers of two Attempts atshifting the reading frame of the uspa1 gene sequence failed to accountfor the

TABLE VII Summary of the N-terminal Sequences of Internal PeptideFragments Digest Sequence^(a) CNBr AAQAALSGLFVPYSVGKFNATAALGGYGSK SEQ IDNO: 26 GKITKNAARQENG SEQ ID NO: 27 LysC Digest VIGDLGRKV SEQ ID #1 NO:28 ALEXNVEEGL SEQ ID NO: 29 ALESNVEEGLXXLS SEQ ID NO: 30 ALEFNGE SEQ IDNO: 31 LysC Digest SITDLGXKV SEQ ID #2 NO: 32 SITDLGTIVDGFXXX SEQ ID NO:33 SITDLGTIVD SEQ ID NO: 34 Trypsin VDALXTKVNALDXKVNSDXT SEQ ID NO: 35LLAEQQLNGKTLTPV SEQ ID NO: 36 AKHDAASTEKGKMD SEQ ID NO: 37ALESNVEEGLLDLSG SEQ ID NO: 38 Trypsin NQNTLIEKTANK SEQ ID Digest #1 NO:39 IDKNEYSIK SEQ ID NO: 40 SITDLGTK SEQ ID NO: 41 Trypsin NQNTLIEK SEQID Digest #2 NO: 42 ALHEQQLETLTK SEQ ID NO: 43 NSSD SEQ ID NO: 44NKADADASFETLTK SEQ ID NO: 45 FAATAIAKDK SEQ ID NO: 46 KASSENTQNIAK SEQID NO: 47 RLLDQK SEQ ID NO: 48 Chymo- AATADAITKNGX SEQ ID trypsin NO: 49AKAXAANXDR SEQ ID NO: 50 Digest of NQADIAQNQTDIQDLAAYNELQ SEQ IDresearch NO: 51 grade UspA NQADIANNINNIYELAQQQDQ SEQ ID with cys-C- NO:52 endo- YNERQTEAIDALN SEQ ID peptidase NO: 53 ILGDTAIVSNSQD SEQ ID NO:54 ^(a)Certain residues of several peptides could not be verified andthese ambiguities are shown by an “X” in SEQ ID NO: 29, SEQ ID NO: 30,SEQ ID NO: 32, SEQ ID NO: 33, SEQ ID NO: 35, SEQ ID NO: 49 and SEQ IDNO: 50. In SEQ ID NO: 29 the ambiguous residue is likely to be a serin;in SEQ ID NO: 33, position 13 is likely to be aspartic acid, position 14is likely to be # glycine and position 15 is likely to be arginine; inSEQ ID NO: 35 both positions 13 and 19 are likely to be serines; in SEQID NO: 49 the ambiguous residue is likely to be an asparagine; and inSEQ ID NO: 50 position 4 is likely to be serine and position 8 is likelyto be threonine.

Additional attempts to resolve the very high molecular weight UspAprotein band from M. catarrhalis strain O35E by SDS-PAGE, followed byelectroelution and digestion with proleases or with cyanogen bromide,again yielded a number of peptides which were sequenced. Severalpeptides (peptides 1-6, Table VIII) were obtained. The amino acidsequence of which was identical or very similar to that deduced from thenucleotide sequence of the uspA1 gene. However, several additionalpeptides, peptides 7-10, Table VIII, were not present in the dolucedamino acid sequence. This finding substantiated the suggestion that asecond protein was present in the UspA antigen preparation.

TABLE VIII Peptide # Amino acid sequence Matching or closely matchingpeptides: Peptide 1 KALESNVEEGLLDLSGR (SEQ ID NO: 55) Peptide 2ALESNVEEGLLELSGRTIDQR (SEQ ID NO: 56) Peptide 3 NQAHIANNINXIYELAQQQDQK(SEQ ID NO: 57) Peptide 4 NQADIAQNQTDIQDLAAYNELQ (SEQ ID NO: 58) Peptide5 ATHDYNERQTEA (SEQ ID NO: 59) Peptide 6 KASSENTQNIAK (SEQ ID NO: 60)Nonmatching peptides: Peptide 7 MILGDTAIVSNSQDNKTQLKFYK (SEQ ID NO: 61)Peptide 8 AGDTIIPLDDDXXP (SEQ ID NO: 62) Peptide 9 LLHEQQLXGK (SEQ IDNO: 63) Peptide 10 IFFNXG (SEQ ID NO: 64) ^(a)Certain residues ofseveral peptides could not be verified and these ambiguities are shownby an “X” in SEQ ID NO: 57, SEQ ID NO: 62, SEQ ID NO: 63 and SEQ ID NO:64.

Further evidence corroborating the assertion that the high molecularweight UspA proteinaceous material was either a multimer of more thanone distinct protein or distinct multimers of two different proteins wasderived from earlier electrospray mass spectroscopic analysis whichpredicted that a monomer of the UspA material had a molecular weight of59,500. This approximately 60 kDa protein reacted immunogenically withthe MAbs 17C7, 45-2, 13-1, and 29-31, in contrast to the UspA1 proteinwhich only cross-reacted with MAb 17C7. The fact that MAb 17C7 reactedwith both isolated proteins suggested that this Mab recognized anepitope common to both proteins.

Preparation of mutant uspA1 construct. The nucleotide sequence of thecloned uspA1 gene was used to construct an isogenic uspA1 mutant.Oligonucleotide primers (BamHI-ended P1 and P16 in Table IX) were usedto amplify a truncated version of the uspA1 ORF from M. catarrhalisstrain O35E chromosomal DNA; this PCR™ product was cloned into the BamHIsite of the plasmid vector pBluescript II SK+. A 0.6 kb BglII fragmentfrom the middle of this cloned fragment was excised and was replaced bya BamHI-ended cassette encoding kanamycin resistance. This new plasmidwas grown in E. coli DH5α, purified by column chromatography, linearizedby digestion with EcoRI, precipitated, and then dissolved in water. Thislinear DNA molecule was used to electroporate the wild-type M.catarrhalis strain O35E, using a technique described previously(Helminen et al., 1993b). Approximately 5,000 kanamycin-resistantlinearized by digestion with EcoRI, precipitated, and then dissolved inwater. This linear DNA 17C7. One of these kanamycin-resistantclones wasrandomly chosen for further examination and Southern blot analysisconfirmed that this mutant was isogenic.

Analysis of products expressed by the uspA1 mutant. When whole celllysates of both the wild-type M. catarrhalis strain and this mutant weresubjected to SDS-PAGE, both the wild-type strain and the mutant strainstill expressed the very high-molecular-weight band originallydesignated as UspA. However, a protein of approximately 120 kDa wasfound to be missing in the mutant strain (FIG. 2A). The fact that boththis mutant and the wild-type parent strain still expressed a very highmolecular weight antigen reactive with MAb 17C7 (FIG. 2B) indicated thatthere had to be a second gene in M. catarrhalis strain O35E that encodeda MAb 17C7-reactive antigen. Furthermore, it should be noted thatEDTA-extracted outer membrane vesicles of both the wild-type strain(FIG. 2C, lanes 5 and 7) and mutant strain (FIG. 2C, lanes 6 and 8)possessed a protein of approximately 70-80 kDa that was reactive withMAb 17C7. This approximately 70-80 kDa band likely represents one form,perhaps the monomeric form, of the product of a second gene encoding theMAb 17C7-reactive epitope.

It is important to note that, when chromosomal DNA from both thewild-type parent strain and the mutant were digested with PvuII andprobed in Southern blot analysis with a 0.6 kb BglII-PvuII fragmentderived from the uspA1 gene, the wild-type strain exhibited a 2.6 kbband and a 2.8 kb band which bound this probe (FIG. 3). In contrast, themutant strain had a 2.6 kb band and a 3.4 kb band that bound this probe.The presence of the 3.4 kb band was the result of the insertion of thekan cartridge into the deletion site in the uspA1 gene.

Example III Characterization of UspA2 and uspA2

Construction of fision proteins. The epitope which binds MAb 17C7 waslocalized by using the nucleotide sequence of the uspa1 gene describedabove to construct fusion proteins. First, fusion proteins containingfive peptides spanning the UspA1 protein were constructed by using thepGEX4T-2 protein fusion system (Pharmacia LKB). The oligonucleotideprimers using the nucleotide sequence of the uspa1 gene described aboveto construct fusion proteins. chromosomal DNA are listed in Table IX.Each of these had either a BamHI site or a XhoI site at the 5′ end,thereby allowing directional in-frame cloning of the amplified productinto the BamHI- and XhoI-digested vector. When recombinant E. colistrains expressing each of ltese five fusion proteins were used in acolony blot radioimmunoassay, only fusion protein MF-4 readily bound MAb17C7. Further analysis of the uspA1-derived nuclcotide sequence in theMF4 fusion construct involved the production of fusion proteinscontaining 79 amino acid residues (MF-4-1) and 123 amino acid residues(MF4-2) derived from the MF-4 fusion protein (Table IX). These twofusion proteins both bound MAb 17C7 (Table IX). FIG. 4 depicts thewestern blot reactivity of MAb 17C7 with the MF4-1 fusion protein. Thesetwo fusion proteins had in common only a 23-residue regionNNINNIYELAQQQDQHSSDIKTL (SEQ ID NO:65), suggesting that this 23-residueregion, designated as the “3Q” peptide, contains the epitope that bindsMAb 17C7.

TABLE IX PCR ™ primers used for the production of usp A1 gene fragmentsfor use in the construction of fusion proteins and mutagenesis and thereactivity of the resulting fusion protein with MAb 17C7 ReactivityFragment Generated: Primer Pair^(a) with MAb 17C7 MF-3   P5-P8 − MF-4   P6-P13 + MF-4.1  P7-P12 + MF-4.2 P11-P13 + ^(a)primer sequences are asfollows: P5 GGTGCAGGTCAGATCAGTGAC SEQ ID NO: 66 P6 GCCACCAACCAAGCTGACSEQ ID NO: 67 P7 AGCGGTCGCCTGCTTGATCAG SEQ ID NO: 68 P8CTGATCAAGCAGGCGACCGCT SEQ ID NO: 69 P11 CAAGATCTGGCCGCTTACAA SEQ ID NO:70 P12 TTGTAAGCGGCCAGATCTTG SEQ ID NO: 71 P13 TGCATGAGCCGCAAACCC SEQ IDNO: 72

Elucidation of the MAb 17C7 Epitope. It is important to note that thenucleotide sequence encoding this 23-residue polypeptide (i.e., the 3Qpeptide) was present in the 0.6 kb BglII-PvuII fragment used in theSouthern blot analysis described in Example II. This finding suggestedthat the epitope that bound MAb 17C7 might be encoded by DNA present inboth the 2.6 and 2.8 kb PvuII fragments, with the 2.8 kb PvuII fragmentbeing derived from the cloned uspA1 gene and the 2.6 kb PvuII fragmentrepresenting all or part of another gene encoding this same epitope.

A ligation-based PCR™ system was used to verify this finding.Chromosomal DNA from the mutant strain was digested to completion withPvuII and was resolved by agarose gel electrophoresis. Fragments rangingin size from 2-3 kb were excised from the agarose, blunt-ended, andligated into the EcoRV site in pBluescript II SK+. This ligationreaction mixture was precipitated and used in a PCR™ amplificationreaction. Each PCR™ reaction contained either the T3 or T7 primerderived from the DNA encoding the 3Q peptide. This approach yielded a1.7 kb product with the T3 and P10 primers and a 0.9 kb product from theT7 and P9 primers (FIG. 5). The sum of these two bands is the same asthe 2.6 kb size of the desired DNA fragment.

Nucleotide sequence analysis of these two PCR™ products revealed twoincomplete ORFs which, when joined at the region encoding the 3Qpeptide, formed a 1,728-bp ORF encoding a protein with a calculatedmolecular weight of 62,483 daltons (SEQ ID NO:3). The amino acid

Nucleotide sequence analysis of these two PCR™ products revealed twoincomplete ORFs region extending from amino acids 278-411 in this secondprotein, designated UspA2, was nearly identical to the region in UspA1between amino acids 505-638 (SEQ ID NO:1). Furthermore, these tworegions both contain the 23-mer (the 3Q peptide) that likely containsthe epitope that binds MAb 17C7. It should also be noted that the fourpeptides from Table IX (Peptides 7-10) that were not found in UspA1 werefound to be identical or very similar to peptides in the deduced aminoacid sequence of UspA2. In addition, the first six peptides listed inTable IX, which matched or were very similar to peptides in the deducedamino acid sequence of UspA1, also matched peptides found in the deducedamino acid sequence of UspA2.

Oligonucleotide primers P1 and P2 (Table IX) were used to amplify a2.5-2.6 kb fragment from M. catarrhalis strain O35E chromosomal DNA.Nucleotide sequence analysis of this PCR™ product was used to confirmthe nucleotide sequence of the uspA2 ORF determined from theligation-based PCR™ study. These results proved that M. catarrhalisstrain O35E contains two different ORFs (i.e., uspA1 and uspA2) whichencode the same peptide (i.e., the 3Q peptide) which likely binds MAb17C7. This 3Q peptide appears twice in UspA1 and once in UspA2 (SEQ IDNO:1 and SEQ ID NO:3).

The nucleotide sequences of the two DNA segments encoding these 3Qpeptides in uspA1 are nearly identical, with three nucleotides beingdifferent. These nucleotide differences did not cause a change in theamino acid sequence. The nucleotide sequence of the DNA segment encodingthe 3Q peptide in uspA2 is identical to the DNA encoding the first 3Qpeptide in UspA1.

As seen in FIG. 2C, lane 7, the three dominant MAb 17C7-reactive bandspresent in M. catarrhalis strain O35E outer membrane vesicles haveapparent molecular weights of greater than 200 kDa, approximately 120kDa, and approximately 70-80 kDa. It should be noted that the existenceof several MAb 17C7-reactive bands, with apparent molecular weights ofgreater than 200 kDa, approximately 120 kDa, and approximately 70-80 kDawas also apparent in U.S. Pat. No. 5,552,146 (FIG. 1, lane H).Therefore, the existence of at least more than one M. catarrhalisantigens reactive with MAb 17C7 was apparent as early as 1991. It is nowapparent that the approximately 120 kDa band likely represents themonomeric form of the UspA1 antigen and the approximately 70-80 kDa bandlikely represents the monomeric form of the UspA2 antigen from M.catarrhalis strain O35E. One or more than one of these species mayaggregate to form the very high molecular weight proteinaceous material(i.e. greater than 200 kDa) of the UspA antigen.

A new M. catarrhalis strain O35E genomic library was constructed in thebacteriophage vector ZAP Express (Stratagene, La Jolla, Calif.).Chromosomal DNA from this strain was partially digested with Sau3A1 and4-9 kb DNA fragments were ligated into the vector arms according to theinstructions obtained from the manufacturer. This library was amplifiedin E. coli MRF′. An aliquot of this library was diluted and plated andthe resultant plaques were screened for reactivity with MAb 17C7.Approximately 24 plaques which bound this MAb were detected, theresponsible recombinant bacteriophage were purified by the single plaqueisolation method, and the DNA insert from one of these bacteriophage wassubjected to nucleotide sequence analysis. Nucleotide sequence of the2.6 kb DNA fragment present in this recombinant bacteriophage revealedthat, on one end, it contained an incomplete ORF that encoded the 3Qpeptide. Until its truncation by the vector cloning site, the sequenceof this incomplete ORF was identical or nearly identical to that of theuspA2 ORF derived from the ligation-based PCR™ study describedimmediately above, providing further evidence that two genes which sharea common epitope encode the UspA antigen.

Example IV Purification of and Immunological Properties of the ProteinsUspA1 and UspA2 Materials and Methods

Bacteria. TTA24 and O35E isolates were as previously described inExample I. Additional isolates were obtained from the University ofRochester and the American Type Culture Collection (ATCC). The bacteriawere routinely passaged on Mueller-Hinton agar (Difco, Detroit, Mich.)incubated at 35° C. with 5% carbon dioxide. The bacteria used for thepurification of the protein were grown in sterile broth containing 10 gcasamino acids (Difco Detroit, Mich.) and 15 g yeast extract (BBL,Cockeysville, Md.) per liter. The isolates were stored at −70° C. InMueller-Hinton broth containing 40% glycerol.

Purification of UspA2. Bacterial cells (˜400 g wet wt. of M. catarrhalisO35E) were washed twice with 2 liters of pH 6.0, 0.03 M sodium phosphate(NaPO4) containing 1.0% Triton® X-100 (TX-100) (J.T. Baker Inc.,Philipsburg, N.J.) (pH 6.0) by stirring at room

Purification of UspA2. Bacterial cells (˜400 g wet wt. of M. catarrhalisO35E) were 13,700×g for 30 min at 4° C. Following centrifugation, thepellet was resuspended in 2 liters of pH 8.0, 0.03 MTris(hydroxymethyl)aminomethane-HCl (Tris-HCl) containing 1.0% TX-100and stirred overnight at 4° C. to extract the UspA2 protein. Cells werepelleted by centrifugation at 13,700×g for 30 min at 4° C. Thesupernatant, containing the UspA2 protein, was collected and furtherclarified by sequential microfiltration through a 0.8 gm membrane (CN.8,Nalge, Rochester, N.Y.) then a 0.45 μm membrane (cellulose acetate, lowprotein binding, Corning, Corning, N.Y.).

The entire filtered crude extract preparation was loaded onto a 50×217mm (˜200 ml) TMAE column [650(S), 0.025-0.4 mm, EM Separations,Gibbstown, N.J.] equilibrated with pH 8.0, 0.03 M Tris-HCl buffercontaining 0.1% TX-100 (THT). The column was washed with 400 ml ofequilibration buffer followed by 600 ml of 0.25 M NaCl in 0.03 M THT.UspA2 was subsequently eluted with 800 ml of 1.0 M NaCl in 0.03 M THT.Fractions were screened for UspA2 by SDS-PAGE and pooled. Pooledfractions (˜750 ml), containing UspA2, were concentrated approximatelytwo-fold by ultrafiltration using an Amicon stirred cell (Amicon Corp.,Beverly, Mass.) with a YM-100 membrane under nitrogen pressure. The TMAEconcentrate was split into two 175 ml aliquots and each aliquot bufferexchanged by passage over a 50×280 mm (˜550 ml) Sephadex G-25 (Coarse)column (Pharmacia Biotech, Piscataway, N.J.) equilibrated with pH 7.0,10 mM NaPO₄ containing 0.1% TX-100 (10 mM PT). The buffer exchangedmaterial was subsequently loaded onto a 50×217 mm (˜425 ml) ceramichydroxyapatite column (Type I, 40 μm, Bio-Rad) equilibrated with 10 mMPT. The column was washed with 450 ml of the equilibration bufferfollowed by 900 ml of pH 7.0, 0.1M NaPO₄ containing 0.1% TX-100. UspA2was then eluted with a linear pH 7.0 NaPO₄ concentration gradientbetween 0.1 and 0.2 M NaPO₄ containing 0.1% TX-100. An additional volumeof pH 7.0, 0.2 M NaPO₄ containing 0.1% TX-100 was applied to the columnand collected to maximize the recovery of UspA2. Fractions were screenedfor UspA2 by SDS-PAGE and pooled. The column was then washed with 900 mlof pH 7.0, 0.5 M NaPO₄ containing 0.1% TX-100. The fractions from thiswash were screened for UspA1 by SDS-PAGE, pooled, and stored at 4° C.This pool was used for the purification of UspA1.

Purification of UspA1. The UspA1 enriched fractions collected duringfour separate purifications of UspA2 were pooled. The combined UspA1pools were concentrated approximately threefold by ultrafiltration usingan Amicon stirred cell with a YM-100 membrane under nitrogen pressure.The UspA1 concentrate was split into two 175 ml aliquots and the bufferexchanged by passage over a 50×280 mm (˜550 ml) Sephadex G-25 columnequilibrated with 10 mM PT. The buffer exchanged material wassubsequently loaded onto a 50×217 mm (˜425 ml) ceramic hydroxyapatitecolumn (Bio-Rad) equilibrated with 10 mM and the buffer exchanged bypassage over a 50×280 mm (˜550 ml) Sephadex G-25 column 7.0, 0.25 MNaPO₄ containing 0.1% TX-100. UspA1 was subsequently eluted with alinear 50×217 mm (˜425 ml) ceramic hydroxyapatite column (Bio-Rad)equilibrated with 10 mM containing UspA1 were identified by SDS-PAGE andpooled.

SDS-PAGE and Western blot Analysis. SDS-PAGE was carried out asdescribed by Laemmli (1970) using 4 to 20% (w/v) gradient acrylamidegels (Integrated Separation Systems (ISS), Natick, Mass.), Proteins werevisualized by staining the gels with Coomassie Brilliant Blue R250. Gelswere scanned using a Personal Densitometer SI (Molecular Dynamics Inc.,Sunnyvale, Calif.) and molecular weights were estimated with theFragment Analysis software (version 1.1) using the prestained molecularweight markers from ISS as standards. Transfer of proteins topolyvinylidene difluoride (PVDF) membranes was accomplished with asemi-dry electroblotter and electroblot buffers (ISS). The membraneswere probed with protein specific antisera or MAb's followed by goatanti-mouse alkaline phosphatase conjugate as the secondary antibody(BioSource International, Camarillo, Calif.). Western blots weredeveloped with the BCIP/NBT Phosphatase Substrate System (Kirkegaard andPerry Laboratories, Gaithersburg, Md.).

Protein Estimation. Protein concentrations were estimated by the BCAassay (Pierce, Rockford, Ill.), using bovine serum albumin as thestandard.

Enzymatic and Chemical Cleavages of UspA2 and UspA1.

(i) CNBr Cleavage. Approximately 0.3 mg of the purified protein wasprecipitated with 90% (v/v) ethanol and the pellet resuspended in 100 μlof 88% (v/v) formic acid containing 12 M urea. Following resuspension,100 μl of 88% (v/v) formic acid containing 2 M CNBr (Sigma, St. Louis,Mo.) was added and the mixture incubated overnight at room temperaturein the dark.

(ii) Trypsin and Chymotrypsin Cleavage. Approximately 2 mg of thepurified protein was precipitated with 90% (v/v) ethanol and the pelletresuspended in a total volume of 1 ml of phosphate-buffered saline (PBS)containing 0.1% TX-100. This preparation was added directly

(ii) Trypsin and Chymotrypsin Cleavage. Approximately 2 mg of thepurified protein Indianapolis, Ind.). The reaction mixture was incubatedfor 48 h at 37° C.

(iii) Endoproteinase Lys-C Cleavage. Approximately 2 mg of the purifiedprotein was precipitated with 90% (v/v) ethanol and the pelletresuspended in a total volume of 1.0 ml of PBS containing 0.1% TX-100.This preparation was added directly to a vial containing 15 μg ofendoproteinase Lys-C (Boehringer Mannheim). The reaction mixture wasincubated for 48 h at 37° C.

(iv) Separation of Peptides. The above cleavage reaction mixtures werecentrifuged in an Eppendorf centrifuge at 12,000 rpm for 5 min and thesupernatant loaded directly onto a Vydac Protein C4 HPLC column (TheSeparations Group, Hesperia, Calif.). The solvents used were 0.1% (v/v)aqueous trifluoroacetic acid (TFA) [Solvent A] and acetonitrile:H₂O:TFA,80:20:0.1 (v/v/v) [Solvent B] at a flow rate of 1.0 ml/min. Followingthe initial was with Vydac Protein C4 HPLC column (The SeparationsGroup, Hesperia, Calif.). The solvents used and detected by absorbanceat 220 nm. Suitable fractions were collected, dried in a Speed-Vacconcentrator (Jouan Inc., Winchester, Va.) and resuspended in distilledwater. The fractions were separated by SDS-PAGE in 10 to 18% (w/v,acrylamide) gradient gels (ISS) in a Tris-Tricine buffer system(Schägger and von Jagow, 1987). The fractions containing a singlepeptide band were submitted directly for N-terminal sequence analysis.Fractions displaying multiple peptide bands in SDS-PAGE wereelectrophoretically transferred onto a PVDF Tris-Tricine buffer system(Schägger and von Jagow, 1987). The fractions containing a single R-250and the individual bands excised before submitting them for N-terminalsequence analysis (Matsudaira, 1987).

Determination of subunit size. Determination of molecular weight byMatrix Assisted Laser Desorption/Ionization-Time of Flight (MALDI-TOF)mass spectrometry (Hillenkamp and Karas, 1990) was done on a Lasermat2000 Mass Analyzer (Finnigan Mat, Hemel Hempstead, UK) with3,5-dimethoxy-4-hydroxy-cinnamic acid as the matrix. Cold ethanolprecipitation was done on samples containing ≧0.1% (v/v) TX-100 toremove the detergent. The final ethanol concentration was 90% (v/v). Theprecipitated protein was resuspended in water.

Determination of aggregate sizes by gel filtration chromatogaphy.Approximately 1 mg of the purified protein was precipitated with 90%(v/v) ethanol and the pellet resuspended in a total volume of 1.0 ml ofPBS containing 0.1% TX-100. Two hundred microliters of the preparationwere applied to a Superose-6 HR 10/30 gel filtration column (10×30 mm,Pharmacia) equilibrated in PBS/0.1% TX-100 at a flow rate of 0.5 ml/min.The column was calibrated using the HMW Calibration Kit (Pharmacia)which contains aldolase with a size of 158,000, catalase with a size of232,000; ferritin with a size of 440,000; thyroglobulin with aPharmacia) equilibrated in PBS/0.1% TX-100 at a flow rate of 0.5 ml/min.The column was

Amino Acid Sequence Analysis. N-terminal sequence analysis was carriedout using an Applied Biosystems Model 477A Protein/Peptide Sequencerequipped with an on-line Model 120A PTH Analyzer (Applied Biosystems,Foster City, Calif.). The phenylthiohydantoin (PTH) derivatives wereidentified by reversed-phase HPLC using a Brownlee PTH C-18 column(particle size 5 μm, 2.1 mm i.d.×22 cm 1.; Applied Biosystems).

Immunizations. Female BALB/c mice (Taconic Farms, Germantown, N.Y.), age6-8 weeks, were immunized subcutaneously with two doses of UspA1 orUspA2 four weeks apart. To prepare the vaccine, purified UspA1 or UspA2was added to aluminum phosphate, and the mixture rotated overnight at 4°C. 3-O-deacylated monophosphoryl lipid A (MPL) (Ribi ImmunoChemResearch, Inc.) was added just prior to administration. Each dose ofvaccine contained 5 μg of purified protein, 100 μg of aluminum phosphateand 50 μg of MPL resuspended in a 200 μl volume. Control mice wereinjected with 5 μg of CRM₁₉₇ with the same adjuvants. Serum samples werecollected before the first vaccination and two weeks after the secondimmunization. Mice were housed in a specific-pathogen free facility andprovided water and food ad libitum.

Monoclonal antibodies. The 17C7 MAb was secreted by a hybridoma (ATCCHB11093). MAbs 13-1, 29-31, 45-2, and 6-3 were prepared as previouslydescribed (Chen et al., 1995).

Murine model of M. catarrhalis pulmonary clearance. This model wasperformed as described previously (Chen et al., 1995).

Enzyme linked immunosorbent assay (ELISA) procedures. Two differentELISA procedures were used. One was used to examine the reactivity ofsera to whole bacterial cells and the other the reactivity to thepurified proteins.

For the whole cell ELISA, the bacteria were grown overnight onMueller-Hinton agar and swabbed off the plate into PBS. The turbidity ofthe cells was adjusted to 0.10 at 600 nm and 100 μl added to the wellsof a 96 well Nunc F Immunoplate (Nunc, Roskilde, Denmark). The cellswere dried overnight at 37° C., sealed with a mylar plate sealer andstored at 4° C. until needed. On the day of the assay, the residualprotein binding sites were blocked by adding 5% non-fat dry milk in PBSwith 0.1% Tween 20 (Bovine Lacto Transfer Technique Optimizer [BLOTTO])and incubating 37° C. for one hour. The blocking solution was thenremoved and 100 μl of sera serially diluted in the wells with blotto.The sera were allowed to incubate for 1 h at 37° C. The plate wells weresoaked with 300 ml PBS containing 0.1% Tween 20 for 30 seconds andwashed 3 times for 5 seconds with a Skatron plate washer and thenincubated 1 hr at 37° C. with goat anti-mouse IgG conjugated to alkalinephosphatase (BioSource) diluted 1:1000 in blotto. After washing, theplates were developed at room temperature with 100 μl per well of 1mg/ml p-nitrophenyl phosphate dissolved in diethanolamine buffer.Development was stopped by adding 50 μl of 3N NaOH to each well. Theabsorbance of each well was read at 405 nm and titers calculated bylinear regression. The titer was reported as the inverse of the dilutionextrapolated to an absorption value of 0.10 units.

For the ELISA against the purified proteins, the proteins were dilutedto a concentration of 5 μg/ml in a 50 mM sodium carbonate buffer (pH9.8) containing 0.02% sodium azide (Sigma Chemical Co.). One hundredmicroliters were added to each well of a 96 well E.I.A./R.I.A mediumbinding ELISA plate (Costar Corp., Cambridge, Mass.) and incubated for16 hours at 4° C. The plates were washed and subsequently treated thesame as described for whole cell ELISA procedure.

Complement-dependent bactericidal assay. For this assay, 20 μl of thebacterial suspension containing approximately 1200 cfu bacteria in PBSsupplemented with 0.1 mM CaCl₂:, MgCl₂ and 0.1% gelatin (PCMG) weremixed with 20 μl of serum diluted in PCMG and incubated for 30 min at 4°C. Complement, prepared as previously described (Chen et al., 1996), wasadded to a concentration of 20%, mixed, and incubated 30 min at 35° C.The assay was stopped by diluting with 200 μl of cold, 4° C., PCMG. 50μl of this suspension was spread onto Mueller-Hinton plates. Relativekilling was calculated as the percent reduction in cfu in the samplerelative to that in a sample in which heat inactivated complementreplaced active complement.

Inhibition of bacterial adherence to HEp-2 cells. The effect of specificantibodies on bacterial adherence to HEp-2 cells was examined. A totalof 5×10⁴ HEp-2 cells in 300 μl of RPMI-10 were added to a sterile 8-wellLab-Tek chamber slide (Nunc, Inc., Naperville, Ill.) and incubatedovernight in a 5% CO₂ incubator to obtain a monolayer of cells on theslide. The bacterial adherence to HEp-2 cells was examined. A total of5×10⁴ HEp-2 cells in 300 μl of with a bacterial suspension that had beenincubated with antisera (1:100) at 37° C. for 1 h. The slides were thenwashed with PBS and stained with the Difco quick stain following themanufacturer's instructions. The slide was viewed and photographed usinga light microscope equipped with a camera (Nikon Microphot-SA, Nikon,Tokyo, Japan).

Protein interaction with fibronectin and vitronectin. The interactionsof purified UspA1 and UspA2 with fibronectin were examined by dot blot.Human plasma fibronectin (Sigma Chemical Co., St. Louis, Mo.) wasapplied to a nitrocellulose membrane, and the membrane blocked withblotto for 1 h at room temperature. The blot was then washed with PBSand incubated with purified UspA1 or UspA2 (2 μg/ml in blotto) overnightat 4° C. After three washes with PBS, the membrane was incubated withthe MAb 17C7 diluted in blotto for 2 h at room temperature and then withgoat anti-mouse immunoglobulin conjugated to alkaline phosphatase(BIO-RAD Lab. Hercules, Calif.) (1:2,000 in PBS with 5% dry milk, 2 h,room temperature). The membrane was finally developed with a substratesolution containing nitroblue tetrazolium and 5-bromo-chloro-3-indolylphosphate in 0.1 M tris-HCl buffer (pH temperature). The membrane wasfinally developed with a substrate solution containing

Interaction with vitronectin was examined by a similar procedure. Thepurified UspA1 and UspA2 were spotted onto the nitrocellulose membraneand the membrane blocked with blotto. The membrane was then incubatedsequentially with human plasma vitronectin (GIBCO BRL, Grand Island,N.Y., 1 μg/ml in blotto), rabbit anti-human vitronectin serum (GIBCOBRL), goat anti-rabbit IgG-alkaline phosphatase conjugate and substrate.

Interaction with HEp-2 cells by the purified protein. Each well of a 96well cell culture plate (Costar Corp., Cambridge, Mass.) was seeded with5×10⁴ HEp-2 cells in 0.2 ml RPMI containing 10% fetal calf serum and theplate incubated overnight in a 37° C. incubator

Interaction with HEp-2 cells by the purified protein. Each well of a 96well cell culture plate (Costar Corp., Cambridge, Mass.) was seeded with5×10⁴ HEp-2 cells in 0.2 ml RPMI mouse antisera to either UspA1 or UspA2(1:1000 dilution in PBS containing 5% dry milk), the plate was washedand incubated with rabbit anti-mouse IgG conjugated to horseradishperoxidase (1:5,000 in PBS containing 5% dry milk) (BrookwoodBiomedical, Birmingham, Ala.) at room temperature for 1 h. Finally, theplate was washed and developed with a substrate solution containing2,2′-azino-bis-(3-ethyl-benzthiazoline-6-sulfonic acid) at 0.3 mg/ml inpH 4.0 citrate buffer containing 0.03% hydrogen peroxide (KPL,Gaithersburg, Md.). Whole bacteria of strain O35E were included as apositive control. The highest concentration of the bacteria tested hadan optical density of A₅₅₀=1.0. The abscissa for the bacterial datashown in FIG. 7 plots the values for three fold dilutions of thebacterial suspension.

Results

Purification of UspA1 and UspA2. The inventors developed a large-scale,high yield process for extracting and purifying UspA2 from a pellet ofM. catarrhalis cells. The method consisted of three critical steps.First the UspA2 protein was extracted from the bacteria with pH 8.0,0.03 M THT. Second, the cell extract was applied to a TMAE column andthe UspA2 protein eluted with NaCl. Finally, the enriched fractions fromthe TMAE chromatography were applied to a ceramic hydroxyapatite columnand the UspA2 eluted with a linear NaPO₄ gradient. A yield of 250 mg ofpurified UspA2 was typically obtained from ˜400 g wet weight of M.catarrhalis O35E strain cells. A single band was seen for the UspA2 inSDS-PAGE gels by Coomassie blue staining. It corresponded to a molecularsize of ˜240,000 and contained greater than 95% of the protein based onscanning densitometry (FIG. 6A). A second band reacting with the 17C7MAb at approximately 125,000 could be detected in the UspA2 preparationby western but not by Coomassie blue staining (FIG. 6C). The cells neednot be lysed to achieve this high yield, which suggested this protein ispresent in large amounts on the surface of the bacterium.

A method for the purification of the UspA1 protein was also developed.This protein co-purified with UspA2 through the initial extraction andTMAE chromatography steps. Following hydroxyapatite chromatography,however, UspA1 remained bound to the column and had to be eluted at thehigher salt concentration of 500 mM NaPO₄. The crude UspA1 preparationobtained in this step was reapplied and eluted from the hydroxyapatitecolumn using a linear sodium phosphate gradient. A total of 80 mg ofpurified UspA1 was isolated from ˜1.6 kg wet wt. of M. catarrhalis O35Estrain cells. UspA1 purified using this method migrated at threedifferent apparent sizes on SDS-PAGE depending on the method of samplepreparation. Unheated samples exhibited a single band at ˜280,000,whereas samples heated at 100° C. for 3 min resulted in an apparentmolecular weight shift to ˜350,000. Prolonged healing at 100° C.resulted in a shift of the 350,000 band to one at 100,000 (FIG. 6B).Following heating of the sample for 7 min at 100° C., the band at100,000 contained greater than 95% of the protein based on scanningdensitometry of the Coomassie stained gel. In contrast, UspA2 migratedat 240,000 regardless of the duration of the heating when examined bySDS-PAGE. The different migration behaviors indicated the preparationscontained two distinctly different proteins

Molecular Weight Determinations. MALDI-TOF mass spectrometric analysisfor determination of molecular weight of UspA2 using3,5-dimethoxy-4-hydroxy-cinnamic acid matrix in presence of 70% (v/v)aqueous acetonitrile and 0.1% TFA resulted in the

Molecular Weight Determinations. MALDI-TOF mass spectrometric analysisfor to the expected [M+H]⁺ and [M+2H]²⁺ molecular ions, the [2M+H]⁺ and[3M+H]⁺ ions were also observed. The latter two ions were consistentwith the dimer and the trimer species. Using similar conditions, theinventors were unable to determine the mass of UspA1.

To determine the molecular sizes of the purified proteins in solution,UspA1 and UspA2 were independently run on a Superose-6 HR 10/30 gelfiltration column (optimal separation range: 5,000-5,000,000) calibratedwith molecular weight standards. Purified UspA1 exhibited a nativemolecular size of 1,150,000 and UspA2 a molecular size of 830,000. Thesesizes, however, may be affected by the presence of TX-100.

N-terminal Sequence Analysis of Internal UspA1 and UspA2 Peptides. Allattempts to determine the N-terminal sequences of both UspA and UspA1proved unsuccessful. No sequence could be determined. This suggested twothings. First, the N-terminus of both proteins were blocked, and,second, neither protein preparation contained contaminating proteinsthat were not N-terminally blocked.

Thus, to confirm that the primary sequence of purified UspA1 and UspA2corresponded to that deduced from their respective gene sequences,internal peptide fragments were generated and subjected to N-terminalsequence analysis. Tables X and XI show the N-terminal sequencesobtained for fragments generated from the digestion of the UspA2 andUspA1 proteins, respectively. The sequences matching the primary aminoacid sequence deduced from the respective gene sequences are indicatedfor each fragment. The UspA2 fragments #3 and #4 exhibited sequencesimilarity with residues 505-515 and 605-614 respectively of the aminoacid sequence deduced from the UspA1 gene. In Table XII, UspA1 fragment#3 exhibited sequence similarity with residues #278-294 of the UspA2primary sequence. These sequences corresponded with the domains withinUspA1 and UspA2 that share 93% sequence identity.

The remainder of the sequences, however, were unique to the respectiveproteins.

TABLE X N-terminal sequences of internal UspA2 peptide cleavagefragments UspA2 Fragment Sequence^(a) Match^(b) Cleavage 1) LLAEQQLNGSEQ ID NO: 73  92-100 Trypsin 2) ALESNVEEGL SEQ ID NO: 74 216-225 Lys-C245-254 274-283 3) ALESNVEEGLLDLS SEQ ID NO: 75 274-288 Trypsin*505-515  4) AKASAANTDR SEQ ID NO: 76 378-387 Chymotrypsin *605-614  5)AATAADAITKNGN SEQ ID NO: 77 439-450 Chymotrypsin 6) SITDLGTKVDGFDGR SEQID NO: 78 458-472 Lys-C 7) VDALXTKVNALDXKVN SEQ ID NO: 79 473-488Trypsin 8) AAQAALSGLFVPYSVGKFNATAALGGYGSK SEQ ID NO: 80 506-535 CNBr^(a)Underlined residues denote mismatch with the nucleotide derivedamino acid sequence. Ambiguous residues whose identity could not beverified are denoted by the letter X. ^(b)Asterisk (*) indicates matchwith UspA1. Without asterisk indicates matches with nucleotide derivedamino acid sequence of UspA2.

TABLE XI N-terminal sequences of internal UspA1 peptide cleavagefragments UspA1 Fragment Sequence^(a) Match^(b) Cleavage 1)LENNVEEPXLNLS 456-468 Lys-C 2) DQKADI 473-478 Trypsin 3)NNVEEGLLDLSGRLIDQK 504-521 Lys-C *278-294  4) VAEGFEIF 690-697 Trypsin5) AGIATNKQELILQNDRLNRI 701-720 Lys-C ^(a)As per Table X. X denotes anunidentified amino acid residue. ^(b)Asterisk (*) indicates match withUspA2. Without asterisk indicates matches with nucleotide derived aminoacid sequence of UspA1.

Reactivity of MAbs with UspA1 and UspA2. The western blot analysis ofpurifed UspA1 and UspA2 revealed that both proteins reacted stronglywith the MAb 17C7 described by Helminen et al. (1994) (FIG. 7). Thereactivity of the proteins with other MAbs was also investigated. Thedata in Table XII show that, whether assayed by ELISA or western, theMAbs 13-1, 29-31 and 45-2 only reacted with UspA2, the MAbs 7D7, 29C6,11A6 and 12D5 only reacted with UspA1, while 17C7 and 6-3 reacted withboth UspA1 and UspA2. All the MAbs shown in Table XIII bind to wholebacteria when examined by ELISA. These results indicated that UspA2 wasexposed on the surface of the bacterium.

TABLE XII Summary of reactivity of monoclonal antibodies with purifiedUspA1, UspA2 and whole bacteria of strain O35E Reactivity Whole PurifiedPurified mAb Isotype bacterium^(a) UspA1^(b) UspA2^(b) 13-1 IgG1κ + − +29-31 IgG1λ + − + 45-2 IgG2a + − + 17C7 IgG2a + + + 6-3 IgM + + + 7D7IgG2b + + − 29C6 IgG1 + + − 11A6 IgA + + − 12D5 IgG1 + + −^(a)Determined by whole cell ELISA. ^(b)Determined by ELISA and westernblot.

TABLE XIII Cross-reactivity of antibodies to UspA1 and UspA2 proteinsGeometric mean ELISA titer^(b) to Antiserum to UspA1 UspA2 UspA1^(a)740,642^(c) 10,748^(c) UspA2^(a)  19,120^(d) 37,615^(d) ^(a)Thepreparation of the sera are described in the text. ^(b)ELISA titers arefor total IgG and IgM antibodies for sera pooled from ten mice. ^(c)Thedifference in titer of the anti-UspA1 with the two purified proteins wasstatistically different by the Wilcoxon signed rank test (p = 0.0002).^(d)The difference in titer of the anti-UspA2 with the two purifiedproteins was statistically different by the Wilcoxon signed rank test (p= 0.01).

Immunogenicity and antibody cross-reactivity. Antisera to the purifiedUspA1 and UspA2 proteins were generated in mice. The titers of antigenspecific antibodies (IgG and IgM) as well as the cross-reactiveantibodies in these sera were determined by an ELISA assay using each ofthe purified proteins (Table XIII). Both proteins elicited antibodytiters that were greater against themselves than against theheterologous protein. Thus, the reactivities of both the MAbs (TableXII) as well as the polyclonal antibodies indicate that the proteinspossessed both shared and non-shared B-cell epitopes.

Antibody reactivity to whole bacterial cells and bactericidal activity.Antisera to the UspA1 and UspA2 were assayed by whole cell ELISA againstthe homologous O35E strain and several heterologous isolates (TableXIV). The antibodies to UspA1 and to UspA2 reacted strongest with theO35E strain. The reactivity of the sera toward the heterologous isolatesindicated they bound antibodies elicited by both UspA1 and UspA2.

TABLE XIV ELISA and complement mediated bactericidal titers toward wholebacterial cells of multiple isolates of M. catarrhalis elicited bypurified UspA1 and purified UspA2 Whole cell ELISA^(a) Bactericidaltiter^(b) anti- anti- anti- anti- Isolate UspA1^(a) UspA2^(a) UspA1UspA2 O35E 195,261 133,492 400 800 430-345  12,693  18,217 400 4001230-359  7,873  13,772 400 400 TTA24  14,341  7,770 800 800 ^(a)Titerdetermined for pool of sera from ten mice. The titer of the sera drawnbefore the first immunization was less than 50 for all isolates.^(b)Bactericidal titers were determined as the inverse of the highestserum dilution killing greater than 50% of the bacteria. The titers forthe sera from mice immunized contemporaneously with CRM₁₉₇ were lessthan 100.

The bactericidal activities of the antisera to UspA1 and UspA2 weredetermined against O35E and other isolates as well (Table XIV). Bothsera had bactericidal titers ranging from 400-800 against O35E and thedisease isolates. Anti-CRM₁₉₇ serum, the negative control, as well assera drawn before immunization, had a titers of <100 against all thestrains. These results were consistent with the previous observationthat the epitopes shared by the two proteins are highly conserved amongisolates and the antibodies toward those isolates are bactericidal.

Pulmonary challenge. Inmmunized mice were given a pulmonary challengewith the homologous O35E strain or the heterologous TTA24 strain.Relative to the control mice immunized with CRM₁₉₇, enhanced clearanceof both strains was observed regardless of whether the mice wereimmunized with UspA1 or UspA2 (Table XV). No statistical difference(p>0.05) was seen between the groups of mice inmmunized with UspA1 andwith UspA2.

TABLE XV Pulmonary clearance of M. catarrhalis by mice immunized withpurified UspA1 and UspA2 Challenge % Study Immunogen strainclearance^(a) p^(a) 1 UspA1   O35E 49.0 0.013 UspA2   31.8 0.05  CRM₁₉₇0  — 2 UspA1   TTA24 54.6 0.02  UspA2   66.6  0.0003 CRM₁₉₇ 0  —^(a)Challenge method described in text. Numbers are the percentage ofbacteria cleared from the immunized mice compared to control mice whichwere immunized with CRM₁₉₇.

Interaction of purified proteins with HEp-2 cells. The purified UspA1and UspA2 were tested for their ability to interact with HEp-2 cellmonolayer in a 96-well plate using an ELISA. Protein binding to theHEp-2 cells was detected with a 1:1 mix of the mouse antisera to UspA1and UspA2. Purified UspA1 bound to HEp-2 cells at concentrations above10 ng. A weak binding by the UspA2 was detected at concentrations above100 ng (FIG. 7). The attachment of O35E bacteria to HEp-2 cells was usedas a positive control. This result, plus the data showing that theanti-UspA1 antibodies inhibited attachment of the bacteria to HEp2cells, suggests UspA1 plays an important role in bacterial attachmentwhich also suggested that UspA1 was exposed on the bacterial surface.

Interaction of purified proteins with fibronectin and vitronectin. Thepurified proteins were assayed for their ability to interact withfibronectin and vitronectin by dot blot assays. Human plasma fibronectinimmobilized on a nitrocellulose membrane bound purified UspA1 but notUspA2 (FIG. 8), while UspA2 immobilized on the nitrocellulose membranewas capable of binding vitronectin (FIG. 8). Vitronectin binding by theUspA1 was also detected, but the reactivity was weaker. Collagen (typeIV), porcine mucin (type III), fetuin and heparin were also tested forinteraction with purified UspA1 and purified UspA2, but these did notexhibit detectable binding.

Discussion

Previous UspA purification attempts yielded preparations containingmultiple high molecular weight protein bands by SDS-PAGE and westernblot. Because each of the bands reacted with the “UspA specific” MAb17C7, it was thought they represented multiple forms of the UspA protein(Chen et al., 1996). However, the inventors have discovered that thereare two distinct proteins, UspA1 and UspA2, that share an epitoperecognized by the 17C7 MAb. These two proteins are encoded by differentgenes. This study shows that UspA1 and UspA2 can be separated from oneanother. The isolated proteins had different SDS-PAGE mobilitycharacteristics, different reactivity with a set of monoclonalantibodies, and different internal peptide sequences. The results,however, were consistent with the proteins sharing a portion of theirpeptide sequences, including the MAb 17C7 epitope. The separation of theproteins from one another has allowed the inventors to furtherdemonstrate how the proteins were different as well as examine theirbiochemical, functional, and immunological characteristics.

In solution, the purified proteins appear to be homopolymers of theirrespective subunits held together by strong non-covalent forces. This isindicated by the fact that UspA2 lacks any cysteines and treatment ofboth proteins with reducing agents did not alter their mobilities inSDS-PAGE. Both gene sequences possess leucine zipper motifs that mightmediate coil—coil interactions (O'Shea et al., 1991). Even so, it wassurprising that the non-covalent bonds of both proteins were not onlystrong enough to resist dissociation by the conditions normally usedSDS-PAGE. Both gene sequences possess leucine zipper motifs that mightmediate coil—coil urea (Klingman and Murphy, 1994) and guanidine HCl. Ofthe two proteins, UspA2 appeared to be less tightly aggregated, this wasindicated by the fact that its subunit size of 59,500 Da could bedetermined by mass spectrometry. UspA1, however, was recalcitrant todissociation by all the methods tried, and this may be the reason itssize could not be determined by mass spectrometry. In SDS-PAGE, thedominant UspA2 migrated with an apparent size of 240,000 while a farsmaller portion migrated at about 125,000 and could only be detected bywestern analysis. The mobility of UspA1, however, varied depending onhow long the sample was heated. The smallest form was about 100,000.This was consistent with the size of the gene product missing from theuspA1 mutant but not with the size predicted from the gene sequence of88,000 Da. In solution, both proteins formed larger aggregates thanthose seen by SDS-PAGE. Their sizes, as measured by gel filtrationchromatography, were 1,150,000 and 830,000 for UspA1 and UspA2respectively. If the proteins behave this way in vivo, UspA1 and UspA2likely occur as large molecular complexes on the bacterial surface ofthe bacterium.

The results of the N-terminal amino acid sequence analyses of the UspA2and UspA1 derived peptides (Tables X and XI) were in agreement with theprotein sequences derived from the respective gene sequences. Thisconfirmed that the purified UspA1 and UspA2 proteins were the productsof the respective uspA1 and uspA2 genes. Further, the experimental andtheoretical amino acid compositions of UspA1 and UspA2 were consistent,given the size of the proteins and the accuracy of the amino aciddetermination. There was, however, a discrepancy between the sizedetermined by mass spectrometry of 59,518 and the size indicated fromthe gene sequence for UspA2 of 62,483. This discrepancy suggested thatthis protein either undergoes post-translational processing orproteolytic degradation.

The data also suggest that both proteins are exposed on the bacterialsurface. That at least one of the proteins is exposed is evident fromthe finding that the MAb 17C7 and polyclonal sera react with wholecells. The reactivities of the UspA2 specific monoclonal antibodies13-1, 29-31 and 45-2 with the bacterial cells in the whole cell ELISAprovided evidence that the UspA2 is a surface protein (Table XII). Thereactivities of the UspA1 specific MAbs 7D7, 29C6, 11A6 and 12D5 withthe bacterial cells in the whole cell ELISA provided evidence that theUspA1 is a surface protein (Table XII). Further evidence for surfaceexposure of UspA1 was indicated by the inhibitory effect of theantiserum on bacterial attachment to HEp-2 cells. The sera to the UspA2lacked this activity. Thus, both UspA1 and UspA2 appeared to be surfaceexposed on the bacterium.

Surface exposure of the proteins is probably important for the twoproteins' functions. One function for UspA1 appears to be meditation ofadherence to host tissues. The evidence for this was that UspA1antibodies inhibited bacterial binding to HEp-2 cells and the purifiedprotein itself bound to the cells. The relevance of binding to HEp-2cells is that they are epithelial cells derived from the larynx, acommon site of M. catarrhalis colonization (Schalen et al., 1992). Thisconfirms the inventors findings that mutants that do not express UspA1fail to bind epithelial cells. The inventors' also showed that UspA1binds fibronectin. Fibronectin has been reported to be a host receptorfor other pathogens (Ljungh and Wadström, 1995; Westerlund and Korhonen,1993). Examination of the gene sequence, however, failed to reveal hasbeen reported to be a host receptor for other pathogens (Ljungh andWadström, 1995; (Westerlund and Korhonen, 1993). Thus, it is fairlyclear that UspA1 plays a role in host adherence, possibly via cellassociated fibronectin.

The function of UspA2 is less certain. Antibodies toward it did notblock adherence to the HEp-2 or Chang cell lines, nor did the purifiedprotein bind to those cells. Yet, UspA2 bound vitronectin strongly.Pathogen binding of vitronectin has been linked to host cell adherence(Gomez-Duarte et al., 1997; Limper et al., 1993); however, van Dijk andhis co-workers have reported that vitronectin binding by M. catarrhalismay be used by the bacteria to subvert host defenses (Verdiun et al.,1994). The soluble form of vitronectin, known as complement factor S,regulates formation of the membrane attack complex (Su, 1996). Theysuggest that the binding of vitronectin to the M. catarrhalis surfaceinhibits the formation of the membrane attack complex, rendering thebacteria resistant to the complement dependent killing activity of thesera They have also described two types of human isolates: one thatbinds vitronectin and is resistant to the lytic activity of the serumand the other that does not bind vitronectin and is serum sensitive (Holet al., 1993). It must be noted, however, that vitronectin, like all theextracellular matrix proteins, has many forms and serves multiplefunctions in the host (Preissner, 1991; Seiffert, 1997). Thus, theinteraction of both UspA1 and UspA2 with the extracellular matrixproteins fibronectin and vitronectin may serve the bacterium in waysbeyond subverting host defenses or as receptors for bacterial adhesion.

Even though the two proteins share epitopes and sequences, they havedifferent biochemical activities and likely serve different biologicalfunctions. If an immune response to the respective protein interfereswith its function, it ought to be considered as a vaccine candidate. Theresults of the immunological studies in mice indicated that bothproteins would be good vaccine candidates. Mice immunized with eitherUspA1 or UspA2 developed high antibody titers toward the homologous andheterologous bacterial isolates. Further, the sera from these mice hadcomplement dependent bactericidal activity toward all the isolatestested. In addition, immunized mice exhibited enhanced pulmonaryclearance of the homologous isolate and heterologous isolates. It isimportant to note that antibodies elicited by the proteins werepartially cross-reactive. This was expected since both react with the17C7 MAb and share amino acid sequence.

Example V The Level and Bactericidal Capacity of Child and Adult HumanAntibodies Directed Against the Proteins UspA1 and UspA2

To determine if humans have naturally acquired antibodies to the UspA1and UspA2 of the M. catarrhalis and the biological activity of theseantibodies if present, sera from healthy humans of various ages wasexamined using both ELISA and a bactericidal assay. It was found thathealthy people have naturally acquired antibodies to both UspA1 andUspA2 in their sera, and the level of these antibodies and theirbactericidal capacity were age-dependent. These results also indicatethat naturally acquired antibodies to UspA1 and UspA2 are biologicallyfunctional, and thus support their use as vaccine candidates to preventM. catarrhalis disease.

Material and Methods

Bacteria. The M. catarrhalis strains O35E and TTA24 were as described inExample I. An ATCC strain (ATCC 25238) and three other clinical isolatesfrom the inventors' collection

Material and Methods

Human sera. Fifty-eight serum samples were collected from a group of tenchildren at 2, 4, 6, 7, 15 and 18 months of age who had received routinechildhood immunizations.

Individual sera from twenty-six adults and fifteen additional children18-36 months of age were also assayed. All sera were obtained fromclinically healthy individuals. Information on M. catarrhaliscolonization and infection of these subjects was not collected. The serawere stored at −70° C.

Purification of UspA1 and UspA2. Purified UspA1 and UspA2 were made fromthe O35E strain of M. catarrhalis as described in Example IV herein.Each protein preparation contained greater than 95% of the specificprotein based on densitometric scanning of Coomassie brilliant bluestained SDS-PAGE. Based on western blot analysis using monoclonalantibodies, each purified protein contained no detectable contaminationof the other.

Purification of UspA1 and UspA specific antibodies from human plasma.Human plasmas from two healthy adults were obtained from the AmericanRed Cross (Rochester, N.Y.) and pooled. The antibodies were precipitatedby adding ammonium sulfate to 50% saturation. The precipitate wascollected by centrifuigation and dialyzed against PBS. A nitrocellulosemembrane (2×3 inches) was incubated with UspA1 or UspA2 at 0.5 mg/ml inPBS containing 0.1% (vol/vol) Triton X-100 for 1 h at room temperature,washed twice with PBS and residual binding sites on the membrane blockedwith 5% (wt/vol) dry milk in PBS for 2 h at room temperature. Themembrane was then sequentially washed twice with PBS, 100 mM glycine (pH2.5) and finally with PBS before incubation with the dialyzed antibodypreparation. After incubating for 4 h at 4° C., the membrane was washedagain with PBS, and then 10 mM Tris buffer (pH 8.0) containing 1 Msodium chloride to remove non-specific proteins. The bound antibodieswere eluted by incubation in 5 ml of 100 mM glycine (pH 2.5) for 2 minwith shaking. One ml of Tris-HCl (1M, pH 8.0) was immediately added tothe eluate to neutralize the pH. The eluted antibodies were dialyzedagainst PBS and stored at −20° C.

Enzyme-linked immunosorbent assay (ELISA). Antibody titers to the O35Eand other M. catarrhalis strains were determined by a whole-cell ELISAas previously described using biotin-labeled rabbit anti-human IgG orIgA antibodies (Brookwood Biomedical, Birmingham, Ala.) (Chen et al.,1996). Antibody titers to UspA1 and UspA2 were determined by a similarmethod except that the plates were coated with 0.1 μg of purifiedprotein in 100 μg of PBS per well overnight at room temperature. The IgGsubclass antibodies to UspA1 or UspA2 were determined using sheepanti-human IgG subclass antibodies conjugated to alkaline phosphatase(The Binding Site Ltd., San Diego, Calif.). The antibody end point titerwas defined as the highest serum dilution giving an A₄₁₅ greater thanthree times that of the control. The control wells received alltreatments except human sera and usually had absorbance values rangingfrom 0.03 to 0.06.

The specificity of biotin-labeled rabbit anti-human IgG and IgAantibodies was determined against purified human IgG, IgM and IgA(Pierce, Rockford, Ill.) by ELISA. No cross-reactivity was found. Theassay sensitivity determined by testing against purified humanantibodies of appropriate isotype in an ELISA was 15 and 60 ng/ml in theIgG and IgA assays, respectively. Likewise, the specificity of the humanIgG subclass antibody assays was confirmed in ELISA against purifiedhuman myeloma IgG subclass proteins (ICN Biomedicals, Inc., Irvine,Calif.), and the assay sensitivity was 15 ng/ml in the IgG1, IgG3 andIgG4 assays, and 120 ng/ml in the IgG2 assay. Two control sera wereincluded to control for assay to assay variation.

Comnplement dependent bactericidal assay. The bactericidal activity ofthe human sera was determined as described previously (Chen et al.,1996). In some studies, the sera were absorbed with purified UspA1 orUspA2 prior to the assay. The absorption of specific antibodies fromthese sera was accomplished by adding the purified proteins to 20 or 50μg/ml final concentration. The final serum dilution was 1:10. Themixtures were incubated for 2 h at 4° C. and the precipitate removed bymicro-centrifugation. The purified human antibodies specific for UspA1and UspA2 were assayed against five M. catarrhalis strains in a similarmanner.

Statistics. Statistical analysis was performed on logarithmictransformed titers using JMP software (SAS institute, Cary, N.C.). Toallow transformation, a value of one half the lowest serum dilution wasassigned to sera which contained no detectable titers. Comparison of IgGlevels among the age groups was done by analysis of variance, and therelationship of antibody titer and the bactericidal titer was determinedby logistic regression. A p value less than 0.05 was consideredsignificant.

Results

Comparison of serum IgG and IgA titers to UspA1 and UspA2 in childrenand adults. The IgG and IgA antibody titers in the sera from tenchildren collected longitudinally between 2-18 months of age, as well asthe random samples from fifteen 18-36 month old children and twenty-sixadults were determined against the whole bacterial cells of the O35Estrain, the purified UspA1 and the purified UspA2 by ELISA. IgG titersto all three antigens were detected in almost all the sera (FIG. 9). TheIgG titers to UspA1 and UspA2 exhibited strong age-dependent variationwhen compared to IgG titers to the O35E bacterium (FIG. 9). The adultsera had significantly higher IgG titers to the purified proteins thansera from children of various age groups(p<0.01). Sera from children at6-7 months of age had the lowest IgG titers to UspA proteins and themean titer at this age was significantly lower than that at 2 months ofage (p<0.05).

The level of IgA antibodies to UspA1, UspA2 and O35E bacterial cellswere age dependent (FIG. 9). A serum IgA titer against the UspA1 andUspA2 was detected in all twenty-six adults and children of 18-36 monthsof age. For children less than 18 months of age, the proportionexhibiting antigen specific IgA titers increased with age. The mean IgAliters to UspA1, UspA2 or O35E bacterium in these sera were low for thefirst 7 months of age but gradually increased thereafter (FIG. 9).

Age-depndent subclass distribution of IgG antibodies to UspA1 and UspA2.The IgG subclass titers to the UspA1 and UspA2 antigens were determinedon sera from ten adult sera and thirty-five children's sera. Thesubclass distribution was found to be age-dependent. The most prominentantibodies to the UspA1 and UspA2 antigens were of the IgG1 and IgG3subclasses, which were detected in almost all sera. The IgG2 and IgG4titers were either undetectable or extremely low. Therefore, only dataon IgG1 and IgG3 subclasses are reported (FIG. 10). The IgG3 titersagainst UspA1 or UspA2 in the adult sera were significantly higher thanthe IgG1 titers (p<0.05). The same subclass profile was seen in the serafrom the 2 month old children, although the difference between IgG1 andIgG3 titers did not reach statistical significance, probably because ofthe smaller sample size. Sera from children between 4 and 36 months ofage all had a similar subclass profile which was different from that ofthe adults, and 2 month old children. The IgG1 titers in children's serawere either higher than or equivalent to the IgG3 titers. The mean IgGItiter to either UspA1 or UspA2 was significantly higher than IgG3 titerto the same antigens in these children's sera (p<0.05).

Bactericidal activity. The bactericidal titers of seventeen serarepresenting different age groups were determined (Table XVI). All theadult sera and three out of five sera from the two month old childrenwhich had high IgG titers to the UspA proteins had strong bactericidalactivity. Sera from 6 month old children had the least bactericidalactivity. All five sera form this age group had a marginal bactericidaltiter of 50, the lowest dilution assayed. The bactericidal activity ofthe sera from 18 to 36 month old children was highly variable withtiters ranging from less than 50 to 500. There was a significant linearrelationship between the bactericidal titers and the IgG antibody titersagainst both UspA1 and UspA2 by logistic regression analysis (p<0.01)(FIG. 11).

TABLE XVI The level of IgG antibodies to UspA1 and UspA2 from normalhuman serum and the serum bactericidal activity ELISA IgG titer^(b) BCSubject^(a) Age UspA1 UspA2 titer^(c) 1 2 month 17,127  6,268 500 6month 4,273 1,363  50 15 month   798   250 <50 2 2 month 12,078  12,244 500 6 month 1,357   878  50 18 month 14,041  14,488  200 3 2 month30,283  20,362  500 6 month 1,077 1,947  50 18 month 2,478 1,475 <50 4 2month 2,086   869 <50 6 month   530   802  50 18 month 9,767 8,591 200 52 month 3,233 2,655 <50 6 month 2,246   360  50 18 month 26,693  43,703 500 6 1.5-3 year 4,036 2,686  50 7 1.5-3 year 2,037 1,251  50 8 1.5-3year   341   251 <50 9 1.5-3 year 2,538 1,200 500 10  1.5-3 year  10781,370 500 11  1.5-3 year 1,265   953  50 12  adult 161,750  87,180  45013  adult 873,680  248,290  >1350    14  adult 154,650  146,900  450 15 adult 10,330  7,860  50 16  adult 35,780  31,230  150 17  adult 19,130 132,200  450 ^(a)Three consecutive samples from subjects 1 through 5were collected at the stated ages. ^(b)ELISA end point titers topurified UspA1 or UspA2 from the O35E strain were determined as thehighest serum dilution giving an A₄₁₅ greater than three times thebackground. ^(c)BC titers: bactericidal titer assayed against the O35Estrain. Sera were assayed at 1:50, 100, 200, and 500. Bactericidal titerwas determined as the highest serum dilution resulting in killing of 50%or more of the bacteria relative to the control. Control bacteria wereincubated with test serum and heat inactivated complement serum.

Bactericidal activity of sera absorbed with purified UspA1 or UspA2.Because normal human sera contain antibodies to numerous antigens of M.catarrhalis as indicated by western blot, an absorption method was usedto determine the contribution of UspA1 and UspA2 specific antibodiestowards the bactericidal activity. Six adult sera were absorbed withpurified UspA1 or UspA2, and the change in ELISA reactivity to UspAproteins determined. A reduction in ELISA reactivity was seen for allthe sera after absorption (Table XVII). Further, absorption with oneprotein resulted in a reduction of IgG titers to the other protein.Reduction of UspA2 reactivity was of the same degree regardless ofwhether the absorbent was UspA1 or UspA2. In contrast, there was lessreduction in UspA1 reactivity after absorption with UspA2 than withUspA1 (Table XVII). This indicated that antibodies to UspA1 and UspA2were partially cross-reactive.

TABLE XVII ELISA titer of adult sera before and after absorption^(a)Absorbent #1 #2 #3 #4 #5 #6 IgG titers to UspA1 in sample^(b) saline161,750 873,680 154,650 10,330 35,780 19,130 UspA1  2,450  2,210  3,160 1,650   <500  3,010 UspA2  42,620  90,150  33,570  6,420  3,490  4,130IgG titers to UspA2^(b) saline  87,180 248,290 146,900  7,860 31,23013,200 UspA1  2,800  2,120  2,700  2,220   <500   <500 UspA2   <500 1,820  3,010  2,960   <500   <500 ^(a)Absorption: An aliquot of adultserum was diluted and added with purified UspA1 or UspA2 from O35Estrain to a final 50 μg/ml protein concentration and final 1:10 serumdilution. The mixtures were incubated at 4° C. for 2 h, and precipitatesremoved by microcentrifugation. ^(b)IgG titers against the UspA1 andUspA2 proteins were end point titers determined with a starting serumdilution of 1:500.

The bactericidal titers of the absorbed sera were determined andcompared with those seen before absorption (Table XVIII). Absorptionwith either UspA1 or UspA2 resulted in complete loss of bactericidalactivity (<50) for all six sera when assayed against the O35E strain,the strain from which the purified proteins were made (Table XVIII). Thebactericidal activity of the absorbed sera was also reduced by at leastthree fold when assayed against the a heterologous strain 1230-359.Absorption using UspA1 resulted in greater reduction of the bactericidaltiter against the heterologous strain in 3 out of 6 samples compared toabsorptions using UspA2 (Table XVIII). This result was consistent withthe difference in the reductions of heterologous strain 1230-359.Absorption using UspA1 resulted in greater reduction of the combinedproteins UspA1 and UspA2 did not result in further reduction of thebactericidal activity compared to UspA1 alone. All six human seracontained antibodies to a 74 kDa OMP from M. catarrhalis as determinedby western blot analysis, and absorption using the purified 74 kDaprotein did not affect the bactericidal activity of either the O35Estrain or the 1230-357 strain. This indicated that antibodies to theUspA proteins were the major source of the 74 kDa protein did not affectthe bactericidal activity of either the O35E strain or the 1230-357

TABLE XVIII Bactericidal titer of the adult human sera before and afterabsorption^(a) Adsorbent #1 #2 #3 #4 #5 #6 Bactericidal titer to O35Estrain in sample^(b) saline 450 >1350    450  50 150 450 UspA1 <50  <50 <50 <50 <50 <50 UspA2 <50  150  <50 <50 <50 <50 Bactericidal titer to1230-359 strain^(b) saline 450 4050 >1350   150 150 450 UspA1  50  150 <50 <50  50 150 UspA2 150 1350  450 <50  50  50 ^(a)Sera were the sameas those described in Table XVII. ^(b)Bactericidal titer: Thebactericidal activity was measured against the O35E or 1230-359 strainswith 3-fold diluted sera starting at 1:50. The highest serum dilutionresulting in 50% or greater killing was determined as the bactericidaltiter. The purified UspA1 and UspA2 proteins used for absorption weremade from the O35E strain.

Because only small volumes of the children sera were available,absorption of these sera was done using a mixture of UspA1 and UspA2proteins. Absorption resulted in the complete loss or a significantreduction of bactericidal activity in four out of seven sera (TableXIX). The four sera including three from two month old children all hadan initial bactericidal titer of 200 or greater prior to absorption. Theother three sera, which did not show a change in bactericidal titer uponabsorption, all had a marginal titer of 50 before absorption. Thereduction in ELISA reactivity to the UspA proteins after absorptionconfirmed that the antibody concentration had been reduced. Thissuggested that antibodies specific for the UspA1 and UspA2 proteins inchildren's sera were also a major source of the bactericidal activitytowards M. catarrhalis.

TABLE XIX Bactericidal activity of children's sera before and afterabsorption pooled purified UspA1 and UspA2^(a) Age Unabsorbed serumAbsorbed serum Sample (months) A₄₁₅ ^(b) BC titer^(c) A₄₁₅ ^(b) BCtiter^(c) 1  2 0.84 200 0.29 <50   2  2 0.93 200 0.19 <50   3  2 0.98500 0.38 50 4 18 0.88 200 0.43 50 5 15 0.66  50 0.25 50 6 18 0.62  500.32 50 7 15 0.68  50 0.35 50 ^(a)Absorption: Each serum was absorbedwith a mixture of UspA1 and UspA2 proteins from O35E strain at finalprotein concentration of 200, 50 or 20 μg/ml. The same result was seenfor all three absorptions of each sample. Only the data from the assayusing 20 μg/ml of protein are shown. ^(b)A₄₁₅: The absorbance at 415 nmin ELISA using the mixture of UspA1 and UspA2 as detection antigen. Serawere tested at a 1:300 dilution. ^(c)BC titer: Highest serum dilutionresulting in 50% or greater killing of the O35E strain in the assay.Sera were assayed at dilutions 1:50, 200, and 500.

Affinity purified antibodies to UspA1 and UspA2: To confirm theircross-reactivity and bactericidal activity, antibodies to UspA1 or UspA2from adult plasma were isolated by an affinity purification procedure.The purified antibodies reacted specifically with the UspA1 and theUspA2 proteins but not with non-UspA proteins in the O35E lysates in awestern blot assay. The purified antibodies to one protein also reactedto the other with almost equivalent titer in ELISA (Table XX). Bothantibody preparations exhibited reactivity with five M. catarrhalisstrains in the whole-cell ELISA and bactericidal assay (Table XXI). Thebactericidal titers against all five M. catarrhalis strains rangedbetween 400 and 800, which was equivalent to ELISA (Table XX). Bothantibody preparations exhibited reactivity with five M. catarrhalis

TABLE XX Cross-reactivity of affinity purified human antibodies to UspA1and UspA2 in ELISA IgG titers against^(b) Antibodies purified to^(a)UspA1 UspA2 UsPA1 50,468 20,088 UsPA2 53,106 52,834 ^(a)The antibodieswere purified from plasma pooled from two healthy adults by immuneelution using purified UspA1 or UspA2 from the O35E strain immobilizedon nitrocellulose membrane. ^(b)ELISA end point titers are the highestantibody dilutions giving an A₄₁₅ greater than three times thebackground.

TABLE XXI Whole cell ELISA titer and bactericidal titer of affinitypurified human antibodies to UspA1 and UspA2^(a) Assay Whole cell ELISAtiter^(b) BC titer^(c) strain Ab to UspA1 Ab to UspA2 Ab to UspA1 Ab toUspA2 O35E 12,553 9,939 400 800 ATCC25238 30,843 29,512 400 400 TTA2451,511 57,045 800 800 216:96 31,140 23,109 400 400 1230-359 8,495 16,458800 800 ^(a)The purified antibody preparations were the same asdescribed in Table XX. The specific reactivities of the purifiedantibodies to UspA proteins, but not other outer membrane proteins, wereconfirmed by western blots. ^(b)ELISA end point titers are the highestantibody dilutions giving an A₄₁₅ greater than three times thebackground when assayed against whole bacterial cells. ^(c)BC titer:Highest antibody dilution resulting in 50% or greater killing of thebacterial inoculum in the assay. Antibodies (120 μg/ml) were assayed atdilutions 1:100, 200, 400, and 800.

Discussion

Previous studies examining human antibodies to M. catarrhalis wholecells or outer membrane proteins usually focused on a single age group.Further, the biological function of the antibodies was left largelyundetermined (Chapman et al., 1985), and the antigens eliciting thefunctional antibodies were not identified. Thus, these previous studiesdid not provide information as to the role of naturally acquiredantibodies in protection against M. catarrhalis diseases, nor did theyprovide clear information as to what antigens are suitable for vaccinedevelopment. The data from this study indicate that the IgG antibodiesto UspA1 and UspA2 are present in normal human sera and their levels areage-dependent. These antibodies are an important source of serumbactericidal activity in both children and adults.

These data indicated that most children had serum IgG antibodies to bothUspA1 and UspA2 at two months of age although the level varied fromindividual to individual, and the IgG subclass profile in these infantsera was similar to that in adult sera. The infant sera had bactericidalactivity. The absorption studies suggested that the bulk of thebactericidal antibodies in these sera were directed against the UspA1and the UspA2 proteins. These results suggest that the IgG antibodiesdetected in the two month old children are of maternal origin. This isconsistent with the report that umbilical cord serum contains hightiters of antibodies to an extract of M. catarrhalis whole cells(Ejlertsen et al., 1994b).

Due to the lack of clinical information on the study subjects and smallnumber of subjects examined in this study, it could not be determinedwhether maternal antibodies against UspA, although bactericidal invitro, were protective in young children. However, at two months of agethe children had significantly higher serum IgG titers against the UspAproteins and only a few of these children had a low level of IgAantibodies to M. catarrhalis as compared to children at 15-18 months ofage. If serum IgA reflects prior mucosal exposure to the bacterium, thenmost of the children are not infected by M. catarrhalis in the first fewmonths of age. One of the reasons may be that the maternal antibodiespresent in the young children protect them from infection at this age.This is consistent with the finding that young children seldom carrythis bacterium and do not develop M. catarrhalis disease during thefirst months of life (Ejlertsen et al., 1994a).

Children may become susceptible to M. catarrhalis infection as maternalantibodies wane. In this study, the sera from 6 to 7 month oldchildren.had the lowest level of IgG antibodies to the UspA proteins andbarely detectable bactericidal titers against whole cells of M.catarrhalis. By 15 months of age, nearly all children had serum IgAantibodies to the UspA proteins, and the level of IgA antibodies hadsignificantly increased along with the level of IgG antibodies andbactericidal activity when compared with children of 6 to 7 months ofage. This suggested that these children had been exposed to thebacterium and mounted an antibody response. The fifteen sera from thegroup of 18-36 month old children all had IgG and IgA titers to the UspAproteins and the bactericidal titers varied greatly. The UspA specificIgG antibodies in the older children's sera had differentcharacteristics than the antibodies from the two month old children.First, the IgGI antibody titer was significantly higher than the IgG3titer in children's sera, while the opposite was true for the 2 monthold children (FIG. 10). Second, most sera from 2 month old children hadbactericidal activity, while bactericidal activity was barely detectablein the sera from children of 6 months or older. The low antibody leveland the low serum bactericidal activity seen in children between 6-36months of age is consistent with the epidemiological findings thatchildren of this age group have the highest colonization rate andhighest incidence of M. catarrhalis disease (Bluestone, 1986; Ejlertsenet al., 1994b; Leinonen et al., 1981; Roitt et al., 1985; Ruuskanen andHeikkinen, 1994; Sethi et al., 1995; Teele et al., 1989).

Adults, a population usually resistant to M. catarrhalis infections(Catlin, 1990; al., 1994b; Leinonen et al., 1981; Roitt et al., 1985;Ruuskanen and Heikkinen, 1994; Sethi et UspA proteins as well as higherserum bactericidal activity than children. The bactericidal activity ofthe adult sera was clearly antibody-mediated since immunoglobulindepleted sera had no activity (Chen et al., 1996), and the antibodiespurified from adult plasma exhibited complement dependent bactericidalactivity. The antibodies purified from human sera using UspA1 or UspAfrom a single isolate exhibited killing against multiple strains. Thisresult indicates that humans developed bactericidal antibodies towardthe conserved epitopes of UspA proteins in response to naturalinfections.

In all adult samples, the IgG antibodies were primarily of the IgG1 andIgG3 subclasses with IgG3 being higher. This is consistent with previousreports that the IgG3 subclass is a major constituent of the immuneresponse to M. catarrhalis in adults and children greater than 4 yearsof age, but not in younger children (Carson et al., 1994; Goldblatt etal., 1990). Of the four IgG subclasses in humans, IgG3 constitutes onlya minor component of the total immunoglobulin in serum. However, IgG3antibody has the highest affinity to interact with C1q, the initial stepin the classic complement pathway leading to elimination of thebacterium by both complement-dependent killing and opsono-phagocytosis(Roitt et al., 1985). Since IgG3 antibody is efficiently transferredacross the placenta, it may also confer protective immunity to infants.The data from this study indicate that IgG3 antibody to the UspAproteins is an important component of the immune response to naturalinfection and has ini vitro biological activity.

As clinical information related to M. catarrhalis infection was notcollected for the study subjects, it is unknown how the antibodies toUspA1 or UspA2 were induced. When antibodies made against the UspAproteins in guinea pigs were tested for reactivity with other bacterialspecies, including Pseudomonas aeruginosa, Neisseria meningitidis,Neisseria gonorrhoeae, Bordetella pertussis, Escherichia coli, andnontypable Haemophilus influenzae by western blot, no reactivity wasdetected. This suggests that the antibodies were elicited as a specificresponse to the UspA antigens of M. catarrhalis. This is consistent withthis high colonization rate and the endemic nature of this organism inhuman populations. Since the affinity purified antibodies to the twoUspA proteins were cross-reactive, it could not be determined whetherthe human antibodies were elicited by one or both proteins. It seemedclear that the shared sequence between these two proteins was the maintarget of the bactericidal antibodies.

In summary, this study demonstrated that antibodies to the two UspAproteins are present in nearly all humans regardless of age. The overalllevel and subclass distribution of these antibodies, however, wereage-dependent. IgG antibodies against UspA1 and UspA2 werecross-reactive, and are a major source of serum bactericidal activity inadults. The level of these antibodies and serum bactericidal activityappears to correlate with age-dependent resistance to M. catarrhalisinfection. Since humans make an antibody response to many other M.catarrhalis antigens in addition to UspA1 and UspA2 after naturalinfection, it remains to be determined if immunization with one or bothUspA proteins will confer adequate protection in susceptiblepopulations.

Example VI UspA2 as a Carrier for Oligosaccharides

UspA2 as a Pneumococcal Saccharide Carrier.

This study demonstrates that UspA2 can serve as a carrier for apneumccoccal saccharide. A seven valent pneumococcal polysaccharide wasconjugated to UspA2 by

UspA2 as a Pneumococcal Saccharide Carrier. taken on wk 6. Each mousewas immunized subcutaneously (s.c.) in the abdomen with 1 μgcarbohydrate per dose with aluminum phosphate as the adjuvant. A groupof mice was immunized with the PP7F-CRM. conjugate as a control. Thedata for the sera from the 6 wk bleed are shown in Table XXII, TableXXII, and Table XXIV. The conjugate elicited antibodies against both thepolysaccharide as well as bactericidal antibodies to M. catarrhalis.These results demonstrate that UspA2 can serve a carrier for elicitingantibodies to this pneumococcal saccharide and retain its immunogenicityto UspA2.

TABLE XXII Titers elicited by 7F conjugates to the pneumococcalpolysaccharide 7F Antigen IgG ELISA titer to Pn Ps 7F* PP7F-UspA2 mix<100 PP7F-UspA2 conjugate 9,514 PP7F-CRM conjugate 61,333 *Pool of serafrom five mice.

TABLE XXIII ELISA titers of sera against whole cells of three M.catarrhalis isolates Immunogen Strain Tested Group¹ 035E 430-3451230-359 PP7F-UspA2² mix 51,049 4,407 9,124 PP7F CRM conjugate 56 49 47PP7F UspA2 conjugate 31,111 3,529 8,310 ¹Vaccine group consists of 5Swiss-Webster mice. Each group immunized at wk 0 and wk 3 and serumcollected at wk 6.

TABLE XXIV Complement dependent bactericidal antibodies against three M.catarrhalis isolates Immunogen Strain Tested Group¹ 035E 430-3451230-359 PP7F-UspA2 mix 400 400 400 PP7F CRM conjugate <100 <100 <100PP7F UspA2 conjugate 400 400 200 ¹BC₅₀ titer is highest serum dilutionat which >50% of bacteria were killed as compared to serum from wk 0mice. The most concentrated serum tested was a 1:100 dilution.

UspA2 as an Haemophilus b Oligosaccharide Carrier.

This study demonstrates that UspA2 can serve as a carrier for anHaemophilus influenzae type b oligosaccharide (HbO). An HbO sample(average DP=24) was conjugated to UspA2 by aqueous reductive aminationin the presence of 0.1% Triton X-100. The ratio of the HbO to UspA2 was2:1 by weight. Conjugation was allowed to proceed for 3 days at 35° C.and the conjugate diafiltered using an Amicon 100K cutoff membrane. Theconjugate ratio (mg carbohydrate/mg UspA2) was 0.43:1. The carbohydratewas determined by orcinal assay and HbO to UspA2 was 2:1 by weight.Conjugation was allowed to proceed for 3 days at 35° C. and analysis andfound to be 12.6.

The immunogenicity of the conjugate was examined by immunizingSwiss-Webster mice. The mice were immunized twice on wk 0 and wk 4 with1 μg of carbohydrate. No adjuvant was used with the conjugate, but wasused with UspA2. The sera were pooled and titered. The reactivity towardHbPS by the radioantigen binding assay (RABA) was similar to that seenwhen HbO is conjugated to CRM₁₉₇ (Table XXV). The whole cell titertoward the homologous M. catarrhalis isolate (O35E) was similar to thatseen for non-conjugated UspA2 (Table XXVI), as were the bactericidaltiters (Table XXVII). Thus, when a carbohydrate antigen elicits a RABAtiter less than 0.10 is conjugated to UspA2, it becomes immunogenic.

TABLE XXV Comparison of immunogenicity of HbO conjugated to UspA2 to HbOconjugated to CRM₁₉₇ to Haemophilus b polysaccharide by RadioantigenBinding Assay (RABA) Week HbO-CRM₁₉₇ Hbo-UspA2 0 <0.10 <0.10 3 2.51 2.874 4.46 3.56 6 58.66 18.92

TABLE XXVI Comparison of immunogenicity of HbO-UspA2 conjugate withnon-conjugated UspA2 by ELISA against whole cell of the O35E isolate toM. catarrhalis Week UspA2^(a) Hbo-UspA2 0 <50 <50 4 54,284 17,424 6345,057 561,513 ¹5 μg UspA2 adjuvanted with 500 μg aluminum phosphate.

TABLE XXVII Bactericidal of sera toward two M. catarrhalis isolates.Isolate UspA2^(a) Hbo-UspA2 O35D 4,500 >4,500 345 n.d. 450 ^(a)5 μgUspA2 adjuvanted with 500 μg aluminum phosphate. n.d. = not determined

Example VII Association of Mouse Serum Sensitivity with Expression ofMutant Forms of UspA2

When bacteria are killed in the presence of serum that lack specificantibodies toward them, it is called “serum sensitivity.” In the case ofM. catarrhalis, the mutants lacking an intact UspA2 protein have beenfound to be serum sensitive. These mutants were constructed so that one(O35E.1; refer to Example IX for a description of isolates O35E.1,035E.2 and O35E.12) did not express UspA1, one (O35E.2) did not expressUspA2, and one (O35E.12) did not express either protein based on a lackof reactivity with the 17C7 monoclonal antibody. The O35E.2 and O35E.12,however, expressed a smaller truncated form UspA2 (tUspA2) that reactswith antibodies prepared by immunizing mice with purified UspA2. ThetUspA2 could be detected in a western blot of bacterial lysates usingeither polyclonal anti-UspA2 sera or the MAb 13-1. The size of thesmaller form was consistent with the gene truncation used for theconstruction of the two mutants.

This bactericidal capacity was tested by mixing the non-immune mousesera, a 1:5 dilution of human complement and a suspension of bacteria(Approx. 1000 cfu) in the wells of a microtiter plate. The mouse serawere tested at both a 1:50 and 1:100 dilution. The number of survivingbacteria was then determined by spreading a dilution of this bacterialsuspension on agar growth medium. The killing was considered significantwhen fewer than 50% viable bacteria as cfu's were recovered relative tothe samples without mouse sera. Killing by the non-immune sera was seenonly for the mutants lacking a “complete” UspA2 (Table XXVII).

TABLE XXVIII Bactericidal activity of the pre-immune sera from Balb/cmice Bactericidal Activity of Normal Mutant Proteins Expressed MouseSera 035E UspA1 & UspA2 − 035E.1 UspA2 − O35E.2 UspA1 & tUspA2 + 035E.12tUspA2 +

Example VIII Identification of a Decapeptide Epitope in UspA1 that BindsMAb 17C7

It was clear from the work with different strains of M. catarrhalis andanalyses of their protein sequences of UspA1 that certain epitopicregions must exist which are similar,if not identical, in all of thestrains and provide the basis of the immunogenic response in humans. Inorder to identify such immunogenic epitope(s), peptides spanning theUspA1 region known to contain the binding site for MAb 17C7 wereprepared and examined for their ability to bind to identical, in all ofthe strains and provide the basis of the immunogenic response in humans.In

Specifically, overlapping synthetic decapeptides, as shown in Table XXXand FIG. 12, that were N-terminally bound to a membrane composed ofderivatized cellulose were obtained from Research Genetics Inc.(Huntsville, Ala.). After five washes with PBS-Tween containing 5% (w/v)non-fat dry milk, the membrane was subsequently incubated with MAb 17C7(in the form of hybridoma culture supernatant) overnight at 4° C.Following three washes with PBS-Tween, the membrane was incubatedovernight at 4° C. with gentle rocking with 10⁶ cpm of radioiodinated(specific activity 2×10⁷ cpm/μg protein), affinity-purified goatanti-mouse immunoglobulin. The membrane was then washed as before andexposed to X-ray film (Fuji RX safety film, Fuji Industries, Tokyo,Japan).

TABLE XXIX Decapeptides Used to Identify Binding Site for MAb 17C7PEPTIDE # PEPTIDE SEQUENCE 9 SGRLLDQKAD SEQ ID NO:81 10 QKADIDNNIN SEQID NO:82 11 NNINNIYELA SEQ ID NO:83 12 NNIYELAQQQ SEQ ID NO:84 13YELAQQQDQH SEQ ID NO:18 14 AQQQDQHSSD SEQ ID NO:85 15 QDQHSSDIKT SEQ IDNO:86 16 HSSDIKTLKN SEQ ID NO:87 17 DIKTLKNNVE SEQ ID NO:88 18TLKNNVEEGL SEQ ID NO:89 19 EEGLLDLSGR SEQ ID NO:90 20 LSGRLIDQKA SEQ IDNO:91 21 DQKADIAKNQ SEQ ID NO:92 22 AKNQADIAQN SEQ ID NO:93 23IAQNQTDIQD SEQ ID NO:94 24 DIQDLAAYNE SEQ ID NO:95

It is clear from the dot blot results shown in the autoradiograph (FIG.13) that peptide 13, YELAQQDQH (SEQ ID NO:18) exhibited optimal bindingof MAb 17C7 with peptide 14 (SEQ ID NO:85) exhibiting less than optimalbinding. This same peptide (SEQ ID NO:18) is present in UspA2 whichexplains why both proteins bind to MAb 17C7.

Interestingly,peptide 12 shows no binding and binding by peptides 15, 16, 19, 22, 23 is probably non-specific. Thus, a comparison of peptides12, 13, and 14 yields the conclusion that the 7-mer AQQQDQH (SEQ IDNO:17) is an essential epitope for MAb 17C7 to bind to UspA1 and UspA2.This conclusion is in agreement with the current understanding that animmunogenic epitope may comprise as few as five, six or seven amino acidresidues.

Example IX Phenotypic Effect of Isogenic uspA1 and uspA2 Mutations on M.catarrhalis Strain O35E Materials and Methods

Bacterial strains, plasmids and growth conditions. The bacterial strainsand plasmids used in this study are listed in Table XXX. M. catarrhalisstrains were routinely grown at 37° C. on Brain-Heart Infusion (BHI)agar plates (Difco Laboratories, Detroit, Mich.) In an atmosphere of 95%air-5% CO₂ supplemented, when necessary, with kanamycin (20 μg/ml)(SigmaChemicals Co., St. Louis, Mo.) or chloramphenicol (0.5 μg/ml) (Sigma),or in BHI broth. The BHI broth used to grow M. catarrhalis cells forattachment assays was sterilized by filtration. Escherichia coli strainswere cultured on Luria-Bertani (LB) agar plates (Maniatis et al., 1982)supplemented, when necessary, with ampicillin (100 μg/ml), kanamycin (30μg/ml), or chloramphenicol (30 μg/ml).

TABLE XXX Bacterial Strains and Plasmids Used in this Study Strain orplasmid Description Source or reference M. catarrhalis 035E Wild-typeisolate from Helminen et al., 1994 middle ear fluid O35E.1 Isogenicmutant of O35E Aebi et al., 1997 with a kan cartridge in the uspA1structural gene O35E.2 Isogenic mutant of O35E Aebi et al., 1997 with akan cartridge in the uspA2 structural gene O35E.12 Isogenic mutant ofO35E This study with a kan cartridge in the uspA2 structural gene and acat cartridge in the uspA1 structural gene P-44 Wild-type isolate thatSoto-Hernandez et al., exhibits rapid 1989 hemagglutination P-48Wild-type isolate that Soto-Hernandez et al., exhibits slow 1989hemagglutination Escherichia coli DH5α Host for cloning studiesStratagene Plasmids pBluescript II Cloning vector; Amp^(r) StratagenepUSPA1 pBluescript II SK+ with a Aebi et al., 1997 2.7 kb insertcontaining most of the uspA1 gene of M. catarrhalis strain O35EpUSPA1CAT pUSPA1 with a cat cartridge This study replacing the 0.6 kbBglII fragment of the uspA1 gene

Characterization of outer membrane proteins. Whole cell lysates andouter membrane vesicles of M. catarrhalis strains were prepared asdescribed (Murphy and Loeb, 1989; Patrick et al., 1987). Proteinspresent in these preparations were resolved by SDS-PAGE and detected bystaining with Coomassie blue or by western blot analysis as described(Helminen et al., 1993a).

Monoclonal antibodies (MAbs). MAb 17C7, a murine IgG antibody thatreacts with a conserved epitope of both UspA1 and UspA2 from M.catarrhalis strain O35E, as described in earlier examples herein, wasused for immunologic detection of these proteins. MAb 17C7 seas used inthe form of hybridoma culture supernatant fluid in western blot analysisand in the indirect antibody-accessibility assay. MAb 3F12, an IgG MAbspecific for the major outer membrane protein of Haemophilus ducreyi(Klesney-Tait et al., 1997), was used as a negaitive control in theindirect antibody-accessibility assay.

Molecular cloning methods. Chromosomal DNA of M. catarrhalis strain O35Ewas used as the template in a polymerase chain reaction (PCR™) systemtogether with oligonucleotide primers derived from either just after thestart of the strain O35E uspA1 open reading frame (i.e., P1 in FIG. 14)or just after the end of this open reading frame (i.e. P2 in FIG. 14).These primers were designed to contain a BamHI restriction site at their5′-end. The sequence of these primers was:

P1-5′-CGGGATCCGTGAAGAAAAATGCCGCAGGT-3′ (SEQ ID NO:96);

P2-5′-CGGGATCCCGTCGCAAGCCGATTG-3′ (SEQ ID NO:97).

DNA fragments were amplified using a PTC 100 Programmable ThermalController (MJ Research, Inc., Cambridge, Mass.) and the GeneAmp PCR™kit (Roche Molecular Systems, Inc., Branchburg, N.J.). PCR™ productswere extracted from 0.7% agarose gel slices using the Qiaex GelExtraction Kit (Qiagen, Inc., Chadsworth, Calif.) and digested withBamHI (New England Biolabs, Inc., Beverly, Mass.) for subsequentligation into the BamHI site of pBluescript II SK+ (Stratagene, LaJolla, Calif.). Ligation reactions were performed with overnightincubation at 16° C. using T4 DNA ligase (Gibco BRL, Inc., Gaithersburg,Md.). Competent E. coli DH5α cells were transformed with the ligationreaction mixture according to a standard heat-shock procedure (Sambrooket al., 1989) and the desired recombinants were selected by culturing inthe presence of an appropriate antimicrobial compound. The 1.3 kbchloramphenicol (cat) resistance cartridge was prepared by excision(using BamHI) from pUCΔECAT (Wyeth-Lederle, Rochester, N.Y.). The catcartridge was subsequently ligated into BglII restriction sites locatedin the mid-portion of cloned segment from the uspA1 gene and, aftertransformation of competent E. coli DH5 cells, recombinant clones wereidentified by selection on solidified media containing chloramphenicol.

Transformation of M. catarrhalis. The electroporation method used fortransformation of M. catarrhalis strain O35E has been described indetail (Helminen et al., 1 993b). Briefly, a 30-ml portion of alogarithmic-phase broth culture (10⁹ colony forming units [cfu]/ml) Nvasharvested by centrifugation, washed three times with 10% (v/v) glycerolin distilled water, and resuspended in 100 μl of the same solution. A20-μl portion of these cells was electroporated with 5 μg of linear DNA(i.e., the truncated uspA1 gene containing the cat cartridge) in 5 μl ofwater in a microelectroporation chamber (Cel-Porator Electroporationsystem; Bethesda Research Laboratories, Gaithersburg, Md.) by applying afield strength of 16.2 kV over a distance of 0.15 cm. Followingelectroporation, the cell suspension was transferred to 1 ml of BHIbroth and incubated with shaking at 37° C. for 90 min. Ten 100-μlportions were then spread on BHI agar plates containing the appropriateantimicrobial compound.

Southern blot analysis. Chromosomal DNA purified from wild-type andmutant M. catarrhalis strains strains was digested with either PvuII orHindIII (New England Biolabs) and Southern blot analysis was performedas described (Sambrook et al., 1989). Double-stranded DNA probes werelabeled with ³²P by using the Random Primed DNA Labeling Kit(Boehringer-Mannheim, Indianapolis, Ind.). and Southern blot analysiswas performed as described (Sambrook et al., 1989). strain O35E and itsisogenic mutants were diluted in PBS buffer containing 10% (v/v) ietalbovine serum and 0.025% (w/v) sodium azide (PBS-FBS-A) to density of 110Klett units (ca. 10⁹ cfu/ml) as measured with a Klett-Sumrnmersoncalorimeter (Klett Manufacturing Co., New York, N.Y.). Portions (100 μl)of this suspension were added to 1 ml of MAb 17C7 or MAb 3F12 culturesupernatant. After incubation at 4° C. for one hour with gentleagitation, the bacterial cells were washed once and suspended in 1 ml ofPBS-FBS-A. Affinity-purified goat anti-mouse immunoglobulin,radiolabeled with ¹²⁵I to a specific activity of 10⁸ cpm per μg, wasadded and the mixture was incubated for one hour at 4° C. with gentleagitation. The cells were then washed four times with 1 ml of PBS-FBS-A,suspended in 500 μl of triple detergent (Helminen et al., 1993a) andtransferred to glass tubes. The radioactivity present in each sample wasmeasured by using a gamma counter.

Autoagglutination and hemagglutination assays. The ability of M.catarrhalis strains, to autoagglutinate was assessed using bacterialcells grown overnight on a BHI agar plate. These cells were resuspendedin PBS to a turbidity of 400 Klett units in a glass tube andsubsequently allowed to stand at room temperature for ten minutes atwhich time the turbidity of this suspension was again determined. Rapidand slow autoagglutination were defined as turbidities of less that andgreater than 200 Klett units, respectively, after 10 minutes. Thehemagglutination slide assay using hepariized human group 0 Rh⁺erythrocytes was performed as previously described (Soto-Hernandez etal., 1989).

Serum bactericidal assay. Complement-sufficient normal adult human serumwas prepared by standard methods. Complement inactivation was achievedby heating the serum, for 30 min at 56° C. A M. catarrhalis brothculture in early logarithmic phase was diluted in Veronal-bufferedsaline containing 0.10% (w/v) gelatin (GVBS) to a concentration of1-2×10⁵ cfu/ml, and 20 μl portions were added to 20 μl of native orheat-inactivated normal human serum together with 160 μl ofVeronal-buffered saline containing 5 mM MgCl₂ and 1.5 mM CaCl₂. Thismixture was incubated at 37° C. In a stationary water bath. At time 0and at 15 and 30 min, 10 μl aliquots were removed, suspended in 75 μl ofBHI broth and spread onto prewarmed BHI agar plates.

Adherence assay. A method used to measure adherence of Haemophilusinfulenzae to Chang conjunctival cells in vitro (St. Geme III andFalkow, 1990) was adapted for use with M. catarrhalis. Briefly, 2-3×10⁵HEp-2 cells (ATCC CCL 23) or Chang conjunctival cells (ATCC CCL 20.2)were seeded into each well in a 24-well tissue culture plate(Corning-Costar) and incubated for 24 h before use. A 0.3 ml volume froman antibiotic-free overnight culture of M. catarrhalis was inoculatedinto 10 ml of fresh BHI medium lacking antibiotics and this (ATCC CCL20.2) were seeded into each well in a 24-well tissue culture plate(Corning-Costar) (120 Klett units) with shaking in a gyrotory waterbath. The culture was harvested by centrifugation at 6,000×g at 4-8° C.for 10 min. The supernatant was discarded and a Pasteur pipet was usedto gently resuspend the bacterial cells in 5 ml of pH 7.4phosphate-buffered saline (PBS) or PBS containing 0.15% (w/v) gelatin(PBS-G). The bacterial cells were pipet was used to gently resuspend thebacterial cells in 5 ml of pH 7.4 phosphate-buffered

Portions (25 μl) of this suspension (10⁷ CFU) were inoculated into thewells of a 24-well tissue culture plate containing monolayers of HEp-2or Chang cells. These tissue culture plates were centrifuged for 5 minat 165×g and then incubated for 30 min at 37° C.

Portions (25 μl) of this suspension (10⁷ CFU) were inoculated into thewells of a the epithelial cells were then released from the plasticsupport by adding 200 μl of PBS containing 0.05% trypsin and 0.02% EDTA.This cell suspension was serially diluted in PBS or PBS-G and spreadonto BHI plates to determine the number of viable M. catarrhalispresent. Adherence was expressed as the percentage of bacteria attachedto the human cells relative to the original inoculum added to the well.

Results

Construction of an isogenic M. catarrhalis mutant lacking expression ofboth UspA1 and UspA2. Construction of M. catarrhalis mutants lacking theability to express either UspA1 (mutant strain O35E.1) or UspA2 (mutantstrain O35E.2) has been described in previous examples (Aebi et al.,1997). For constructing a double mutant that lacked expression of bothUspA1 and UspA2, the 0.6 kb BglII fragment of pUSPA1 (FIG. 14A) wasreplaced by a cat cassette, yielding the recombinant plasmid pUSPA1CAT.Using the primers P1 and P2, the 3.2 kb insert of pUSPA1CAT wasamplified by PCR™. This PCR™ product was used to electroporate thekanamycin-resistant uspA2 strain O35E.2 and yielded the cassette,yielding the recombinant plasmid pUSPA1CAT. Using the primers P1 and P2,the 3.2

Southern blot analysis was used to confirm that strains O35E.1, O35E.2,and O35E.12 were isogenic mutants and that allelic exchange had occurredproperly, resulting in replacement of the wild-type uspA1 or uspA2 gene,or both, with the mutated allele. Chromosomal DNA preparations from thewild-type parent strain O35E, the uspA1 mutant O35E.1, the uspA2 mutantO35E.2, and the putative uspA1 uspA2 mutant strain O35E.12 were digestedto completion with PvuII and probed in Southern blot analysis with DNAfragments derived from these two M. catarrhalis genes or with the kancartridge. For probing with the cat cartridge, chromosomal DNA fromstrain O35E.12 was digested with HindIII.

The uspA1-specific DNA probe was obtained by PCR™-based amplification ofM. catarrhalis strain O35E chromosomal DNA using the primers P3 and P4(FIG. 14A). A 500-bp uspA2-specific DNA fragment was amplified from O35Echromosomal DNA by PCR™ with the primers P5 and P6 (FIG. 14B). Use ofthese two gene-specific probes together with the kan and cat cartridgesin Southern blot analysis confirmed that strain O35E.12 was a uspA1uspA2 double mutant.

Characterization of selected proteins expressed by the wild-type andmutant M. catarrhalis strains. Proteins present in outer membranevesicles extracted from the the wild-type and these three mutant strainswere resolved by SDS-PAGE and either stained with Coomassie blue (FIG.15A) or probed with MAb 17C7 in western blot analysis (FIG. 15B). Thewild-type parent strain O35E possessed a very high molecular weight banddetectable by Coomassie blue staining (FIG. 15A, lane 1, closed arrow)that was also similarly abundant in the uspA1 mutant O35E.1 (FIG. 15A,lane 2). The uspA2 mutant O35E.2 (FIG. 15A, lane 3) had a much reducedlevel of expression of a band in this same region of the gel; this bandwas not visible at all in the uspA1 uspA2 double mutant O35E.12 (FIG. 2,panel A, lane 4).

Western blot analysis revealed that the wild-type strain (FIG. 15B,lane 1) expressed abundant amounts of MAb 17C7-reactive antigen, most ofwhich had a very high molecular weight, in excess of 220,000. Thewild-type strain also exhibited discrete antigens with apparentmolecular weights of approximately 120,000 and 85,000 which bound thisMAb (FIG. 15B, lane 1, open and closed arrows, respectively). The uspA1mutant O35E.1 (FIG. 15B, line 2) lacked expression of the 120 kDaantigen, which was proposed to be the monomeric form of UspA1, but stillexpressed the 85 kDa antigen. The amount of very high molecular weightMAb 17C7-reactive antigen expressed by this uspA1 mutant appeared to beequivalent to that expressed by the wild-type strain. The uspA2 mutantO35E.2 (FIG. 15B, lane 3) expressed the 120 kDa antigen but lackedexpression of the 85 kDa antigen which was proposed to be the monomericform of the UspA2 protein. In contrast to the uspA1 mutant, the uspA2mutant had relatively little very high molecular weight antigen reactivewith MAb 17C7. Finally, the uspA1 uspA2 double mutant O35E.12 (FIG. 15B,lane 4) expressed no detectable MAb 17C7-reactive antigens.

Binding of MAb 17C7 to whole cells of the wild-type and mutant strains.The indirect antibody-accessibility assay was used to determine whetherboth UspA1 and UspA2 are exposed on the surface of M. catarrhalis andaccessible to antibody. Whole cells of both the wild-type strain O35Eand the uspA1 mutant O35E.1 bound similar amounts of MAb 17C7 (TableXXXI). This result suggested that UspA2 is expressed on the surface ofM. catarrhalis, or at least on the surface of the uspA1 mutant. TheuspA2 mutant O35E.2 bound substantially less MAb 17C7 than did thewild-type strain, but the level of binding was still at least an orderof magnitude greater than that obtained with an irrelevant IgG Mabdirected against a H. ducreyi outer membrane protein (Table XXXI). Asexpected from the western blot analysis, the uspA1 uspA2 double mutantO35E.12 did not bind MAb 17C7 at a level greater than obtained with thenegative controls involving the H. ducreyi-specific MAb (Table XXXI).

TABLE XXXI Binding of MAb 17C7 to the Surface of Wild-Type and MutantStrains of M. catarrhalis Binding^(a) of Strain MAb 17C7 MAb 3F12b O35E(wild-type) 145,583^(c) 4,924 O35E.1 (uspA1 mutant) 154,119 4,208 O35E.2(uspA2 mutant)  96,721 4,455 O35E.12 (uspA1 uspA2 double mutant)  6,0813,997 ^(a)Counts per min of ¹²⁵I-labeled goat anti-mouse immunoglobulinbound to MAbs attached to the bacterial cell surface, as determined inthe indirect antibody-accessibility assay. ^(b)MAb 3F12, a murine IgGantibody specific for a H. ducreyi outer membrane protein (Klesney-Taitet al., 1997), was included as a negative control. ^(c)The valuesrepresent the mean of two independent studies.

Characterization of the growth autoagglutination, and hemagglutinationproperties of the wild-type and mutant strains. The colony morphology ofthese three mutant strains grown on BHI agar plates did not differ fromthat of the wild-type strain parent strain. Similarly, the rate andextent of growth of all four of these strains in BHI broth were verysimilar if not identical (FIG. 16). In an autoagglutination assayperformed as described in above in the Materials and Methods section ofthis example, all four strains exhibited the same rate ofautoagglutination. Finally, there was no detectable difference betweenthe wild-type parent and the three mutants in a hemagglutination assayusing human group 0 erythrocytes (Soto-Hernandez et al., 1989). Controlhemagglutination studies were performed using a pair of M. catarrhalisisolates (i e., strains P-44 and P48) previously characterized as havingrapid or slow rates, respectively, of hemagglutination (Soto-Hernandezet al., 1989). (Soto-Hernandez et al., 1989). Control hemagglutinationstudies were performed using a pair of M. catarrhalis isolates (i.e.,strains P-44 and P48) previously characterized as having rapid or slowrates, respectively, of hemagglutination (Soto-Hernandez et al., 1989).whether lack of expression of UspA1 or UspA2 affected this adherenceability, the wild-type and the three mutant strains were first used inan attachment assay with Hep-2 cells. In this set of studies, PBS wasused as the diluent for washing the HEp-2 cell monolayers and for serialdilution of the trysinized HEp-2 cell monolayer at the completion of theassay. Both the wild-type strain and the uspA2 mutant O35E.2 exhibitedsimilar levels of attachment to HEp-2 monolayers (Table XXXI). The uspA1mutant O35E.1, however, was less able to adhere to these HEp-2 cells;lack of expression of UspA1 reduced the level of attachment byapproximately six-fold (Table XXXII). The uspA1 uspA2 double mutantO35E.12 exhibited a similarly reduced level of attachment (Table XXXII).

TABLE XXXII Adherence of Wild-Type and Mutant Strains of M. catarrhalisto HEp-2 and Chang Conjunctival Cells in vitro Adherence^(a) to StrainHEp-2 cells^(b) Chang cells^(c) O35E (wild-type) 14.7 ± 4.9 51.4 ± 30.8O35E.1 (uspA1 mutant)  2.4 ± 0.9 (0.006^(d))  0.8 ± 0.5 (0.002^(d))O35E.2 (uspA2 mutant) 19.1 ± 7.0 (0.213^(d)) 55.9 ± 16.7 (0.728^(d))O35E.12  2.3 ± 1.8 (0.011^(d))  0.6 ± 0.2 (0.002^(d)) (uspA1 uspA2double mutant) ^(a)Adherence is expressed as the percentage of theoriginal inoculum that was adherent to the human epithelial cells at theend of the 30 min incubation period. Each number represents the mean(±S.D.) of two independent studies. ^(b)PBS was used for washing of themonolayers and for serial dilutions of adherent M. catarrhalis.^(c)PBS-G was used for washing of the monolayers and for serialdilutions of adherent M. catarrhalis. ^(d)P value when compared to thewild-type strain O35E using the two-tailed Student t-test.

Control studies revealed, however, that M. catarrhalis cells did notsurvive well in the PBS used for washing of the HEp-2 monolayer andserial dilution of the attached M. catarrhalis organisms. When 10⁸ CFUof the wild-type and mutant M. catarrhalis strains were suspended inPBS, serially diluted, and allowed to stand for 30 min on ice, theviable number of bacteria decreased to 10⁷ CFU. In contrast, when PBScontaining 0.15% (w/v) gelatin (PBS-G) was used for this same type ofexperiment, there was no reduction in the viability of these M.catarrhalis strains over the duration of the experiment. When the HEp-2cell-based attachment studies were repeated using PBS-G for washing theHEp-2 cell monolayer and as the diluent, there was only a three-foldreduction in adherence of the uspA1 mutant relative to that obtainedwith the wild-type parent strain. This finding suggested that theoriginal six-fold difference in attachment ability observed between thewild-type and uspA1 mutant strain may have been attributable in part toviability problems caused by the use of the PBS wash and diluent.

Subsequent studies using Chang conjunctival cells as the target forbacterial attachment together with a PBS-G wash and diluent revealed asubstantial difference in the attachment abilities of the wild-typestrain and the uspA1 mutant (Table XXXII). Whereas the wild-type anduspA2 mutant exhibited similar levels of attachment to the Chang cells,the extent of attachment of the uspA1 mutant was nearly two orders ofmagnitude less than that of the wild-type parent strain. The uspA1 uspA2double mutant also exhibited a much reduced level of attachment similarto obtained with the uspA1 mutant (Table XXXII).

Effect of the uspA1 and uspA2 mutations on serum resistance of M.catarrhalis. Similar to the majority of disease isolates of M.catarrhalis (Hol et al., 1993; 1995; Verduin et al., 1994), thewild-type strain O35E was resistant to killing by normal human serum invitro (Helminen et al., 1993b). To examine the effect of the lack ofexpression of UspA1 or UspA2 on serum resistance, the wild-type strainand the three mutant strains were tested in a serum bactericidal assay.Both the wild-type strain (FIG. 17, closed diamonds) and the uspA1mutant (Helminen et al., 1993b). To examine the effect of the lack ofexpression of UspA1 or UspA2 indicating that lack of expression of UspA1did not adversely affect the ability of strain O35E.1 to resist killingby normal human serum. However, both the uspA2 mutant O35E.2 (FIG. 17,closed circles) and the uspA1 uspA2 double mutant O35E.12 (FIG. 17,closed squares), having in common the lack of expression of UspA2, werereadily killed by normal human serum. Heat-based inactivation of thecomplement system present in this normal human serum eliminated theability of this serum to kill these latter two mutants (FIG. 17, opencircles and squares).

All of the compositions and methods disclosed and claimed herein can bemade and executed without undue experimentation in light of the presentdisclosure. While the compositions and methods of this invention havebeen described in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to thecompositions and methods and in the steps or in the sequence of steps ofthe method described herein without departing from the concept, spiritand scope of the invention. More specifically, it will be apparent thatcertain agents which are both chemically and physiologically related maybe substituted for the agents described herein while the same or similarresults would be achieved. All such similar substitutes andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as defined by theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

EPO Appl. Publ. No. 0036776

U.S. Pat. No. 5,552,146

U.S. Pat. No. 5,310,687

U.S. Pat. No. 5,238,808

U.S. Pat. No. 5,221,605

U.S. Pat. No. 4,608,251

U.S. Pat. No. 4,603,102

U.S. Pat. No. 4,601,903

U.S. Pat. No. 4,599,231

U.S. Pat. No. 4,599,230

U.S. Pat. No. 4,596,792

U.S. Pat. No. 4,578,770

U.S. Pat. No. 4,554,101

U.S. Pat. No. 4,452,901

U.S. Pat. No. 4,367,110

U.S. Pat. No. 4,358,535

U.S. Pat. No. 4,196,265

U.S. Pat. No. 4,174,384

U.S. Pat. No. 3,949,064

U.S. Pat. No. 3,791,932

Aebi, Stone, Beucher, Cope, Maciver, Thomas, McCracken Jr., Sparling,Hansen, “Expression of the CopB outer membrane protein by Moraxellacatarrhalis is regulated by iron and affects iron acquisition fromtransferrin and lactoferrin,” Infect. Immun., 64:2024-2030, 1996.

Altschul, Gish, Miller, Myers and Lipman. “Basic local alignment searchtool,” J. Mol. Biol., 215:403-410, 1990.

Baichwal and Sugden, In: GENE TRANSFER Kucherlapati, R., ed. New York:Plenum Press, pp. 117-148, 1986.

Barenkamp and St. Geme III, “Identification of a second family ofhigh-molecular-weight adhesion proteins expressed by nontypableHaemophilus influenzae,” Mol. Microbiol., 19:1215-1223, 1996.

Bartos and Murphy, “Comparison of the outer membrane proteins of 50strains of Branhamella catarrhalis,” J. Infect. Dis., 158:761-765, 1988.

Benz and Schmidt, “Cloning and expression of an adhesion (AIDA-I)involved in diffuse adherence of enteropathic Escherichia coli,” Infect.Immun., 57:1506-1511, 1989.

Benz and Schmidt, “Isolation and serologic characterization of AIDA-I,the adhesion mediating the diffuse adherence phenotype of thediarrhea-associated Escherichia coli strain 2787 (0126:H27),” Infect.Immun., 60:13-18, 1992b.

Berk, Arch. Intern. Med., 150:2254-2257, 1990.

Bliska, Copass, Falkow, “The Yersinia pseudotuberculosis adhesion YadAmediates intimrate bacterial attachment to and entry into HEp-2 cells,”Infect. Immun., 61:3914-3921, 1993.

Bluestone, “Otitis media and sinusitis in children: role of Branhamellacatarrhalis,” Drugs, 31(Suppl. 3):132-141, 1986.

Bluestone, Stephenson, Martin, “Ten-year review of otitis mediapathogens,” Pediatr. Infect. Dis. J., 11:S7-S11, 1992.

Bolivar et al., Gene, 2:95, 1977.

Brutlag et al., CABIOS, 6:237-245, 1990.

Campagnari, Shanks, Dyer, “Growth of Moraxella catarrhalis with humantransferrin and lactoferrin: Expression of iron-repressible proteinswithout siderophore production,” Infect. Immun., 62:4909-4914, 1994.

Catlin, “Branhamella catarrhalis: an organism gaining respect as apathogen,” Clin Microbiol. Rev., 3:293-320, 1990.

Chang et al., Nature, 375:615, 1978.

Chapman et al., J. Infect. Dis., 151:878-882, 1985.

Chen, McMichael, Vandermeid, Hahn, Smith, Eldridge, Cowell, “Antibodiesto the UspA outer membrane protein of Moraxella catarrhalis blockbacterial attachment in vitro and are protective in a murine pulmonarychallenge model,” Abstracts General Meeting Amer. Soc. Microbiol.,E-53:290, 1995.

China, Sory, N'Guyen, de Bruyere, Cornelis, “Role of YadA protein inprevention of osponization of Yersinia enterocolitica by C3b molecules,”Infect. Immun., 61:3129-3136, 1993.

Christensen, Renneberg, Bruun, Forsgren, “Serum antibody response toproteins of Moraxella (Branhamella) catarrhalis in patients with lowerrespiratory tract infection,” Clin. Diagn. Lab. Immunol., 2:14-17, 1995.

Coligan et al. (eds), In: CURRENT PROTOCOLS IN IMMUNOLOGY, John Wiley,New York, ch. 2.5, 1991.

Consensus, Pediater. Infect. Dis. J., 8:S94-S97, 1989.

Davies and Maesen, “The epidemiology of respiratory tract pathogens inSouthern Netherlands,” Eur. Respir. J., 1:415-420, 1988.

Devereux, Haeberli and Smithies, “A comprehensive set of sequenceanalysis programs for the VAX,” Nucleic Acids Res., 12:387-395, 1984.

Doern, Diag. Microbiol. Infect. Dis., 4:191-201, 1986.

Doyle, Peditr. Infect. Dis. J., 8(Suppl):S45-7, 1989.

Faden et al., Ann. Otol. Rhinol. Laryngol., 100:612-615, 1991.

Faden et al., Pediatr. Infect. Dis. J., 9:623-626, 1990.

Faden, “Comparison of the local immune response to nontypeableHaemophilus influenzae (nHI) and Moraxella catarrhalis (MC) duringotitis media,” In: Advances in Mucosal Immunology, J. Mestecky and etal. (ed.), Plemun Press, New York, p. 733-736, 1995.

Faden, Duffy, Wasielewski, Wolf, Krystofik, Tung, Tonawanda/WilliamsburgPediatrics, “Relationship between nasopharyngeal colonization and thedevelopment of otitis media in children,” J. Infect. Dis.,175:1440-1445, 1997.

Faden, Harabuchi, Hong, TonawandalWilliamsburg Pediatrics, “Epidemiologyof Moraxella catarrhalis in children during the first 2 years of life:Relationship to otitis media,” J. Infect. Dis., 169:1312-1317, 1994.

Faden, Hong and Murphy, “Immune response to outer membrane antigens ofMoraxella catarrhalis in children with otitis media,” Infect Immun.,60:3824-3829, 1992.

Fetrow & Bryant, Biotechnology, 11:479-483, 1993.

Fitzgerald, Mulcahy, Murphy, Keane, Coakley, Scott, “A 200 kDa proteinis associated with haemagglutinating isolates of Moraxella(Branhaemella) catarrhalis,” FEMS Immunol. Med. Microbiol., 18:209-216,1997.

Fleischmann, Adams, White, Clayton, Kirkness, Kerlavage, Bult, Tomb,Dougherty, Merrick, McKenney, Sutton, FitzHugh, Fields, Gocayne, Scott,Shirley, Liu, Glodek, Kelley, Weidman, Phillips, Spriggs, Hedblom,Cotton, Utterback, Hanna, Nguyen, Saudek, Brandon, Fine, Frichman,Fuhrmann, Geoghagen, Gnehm, McDonald, Small, Fraser, Smith and Venter,“Whole-genome random sequencing and assembly of Haemophilus influenzaeRd., Science, 269:496-512, 1995.

Gefter et al., Somatic Cell Genet., 3:231-236, 1977.

Gish and States, “Identification of protein coding regions by databasesimilarity search,” Nat. genet., 3:266-272, 1993.

Glorioso et al., Ann. Rev. Microbiol. 49:675-710, 1995.

Goeddel et al., Nature 281:544, 1979.

Goeddel et al., Nucleic Acids Res., 8:4057, 1980.

Goldblatt, Scadding, Lund, Wade, Turner, Pandey, “Association of Gmallotypes with the antibody response to the outer membrane proteins of acommon upper respiratory tract organism, Moraxella catarrhalis,” J.Immunol., 153:5316-5320, 1994.

Goldblatt, Turner, and Levinsky, “Branhamella catarrhalis: antigenicdeterminants and the development of the IgG subclass response inchildhood,” J. Infect. Dis., 162:1128-1135, 1990.

Hager, Verghese, Alvarez, Berk, “Branhamella catarrhalis respiratoryinfections,” Rev. Infect. Dis., 9:1140-1149, 1987.

Helminen, Beach, Maciver, Jarosik, Hansen, Leinonen, “Human immuneresponse against outear membrane proteins of Moraxella (Branhamella)catarrhalis determined by immunoblotting and enzyme immunoassay,” Clin.Diagn. Lab. Immunol., 2:35-39, 1995.

Helminen, Maciver, Latimer, Cope. McCracken Jr., and Hansen, “A majorouter membrane protein of Moraxella catarrhalis is a target forantibodies that enhance pulmonary clearance of the pathogen in an animalmodel,” Infect. Immun., 61:2003-2010, 1993a.

Helminen, Maciver, Latimer, Klesney-Tait, Cope, Paris, McCracken Jr.,and Hansen, “A large, antigenically conserved protein on the surface ofMoraxella catarrhalis is a target for protective antibodies,” J. Infect.Dis., 170:867-872, 1994.

Helminen, Maciver, Latimer, Lumbley, Cope, McCracken,Jr., and Hansen,.“A mutation affecting expression of a major outer membrane protein ofMoraxella catarrhalis alters serum resistance and survival of thisorganism in vivo,” J. Infect. Dis., 168:1194-1201, 1993b.

Hess et al., J. Adv. Enzyme Reg., 7:149, 1968.

Hitzeman et al., J. Biol. Chem., 255:2073, 1980.

Hol, Verduin, Van Dijke, Verhoef, Fleer, van Dijk, “Complementresistance is a virulence factor of Branhamella (Moraxella)catarrhalis,” FEMS Immunol. Med. Microbiol., 11:207-212, 1995.

Hol, Verduin, van Dijke, Verhoef, van Dijk, “Complement resistance inBranhamella (Moraxella) catarrhalis,” Lancet, 341:1281, 1993.

Holland et al., Biochemistry, 17:4900, 1978.

Horstmann, Sievertsen, Knobloch, Fischetti, “Antiphagocytic activity ofstreptococcal M protein: selective binding of complement control proteinfactor H,” Proc. Natl. Acad.

Holland et al., Biochemistry, 17:4900, 1978.

Horstmann, Sievertsen, Knobloch, Fischetti, “Antiphagocytic activity ofstreptococcal M conservation in stains recovered from the humanrespiratory tract,” Microb. Pathog., 19:215-225, 1995.

Itakura et al., Science, 198:1056, 1977.

Jameson and Wolf, Comput. Appl. Biosci., 4(1):181-186, 1988.

Jones, Genetics, 85:12, 1977.

Jordan, Berk, Berk, “A comparison of serum bactericidal activity andphenotypic characteristics of bacteremic, pneumonia-causing strains, andcolonizing strains of Branhamella catarrhalis,” Am. J. Med.,88(5A):28S-32S, 1990.

Kimura, Gulig, McCracken, Loftus and Hansen, “A minorhigh-molecular-weight outer me,brane protein of Haemophilus influenzaetype b is a protective antigen,” Infect. Immun., 47:253-259,1985.Kingsman et al., Gene, 7:141, 1979.

Klesney-Tait, Hiltke, Spinola, Radolf, Hansen, “The major outer membraneprotein of Haemophilus ducreyi consists of two OmpA homologs,” J.Bacteriol., 179:1764-1773, 1997.

Klingman and Murphy, “Purification and characterization of ahigh-molecular-weight outer membrane protein of Moraxella (Branhamella)catarrhalis,” Infect. Immun., 62:1150-1155, 1994.

Klingman and Murphy, “Purification and characterization of ahigh-molecular-weight outer catarrhalis in bronchiectasis,” Am. J.Respir. Crit. Care Med., 152:1072-1078, 1995.

Kohler & Milstein, Eur. J. Immunol., 6:511-519, 1976.

Kohler & Milstein, Nature, 256:495497, 1975.

Kovatch, Wald, Michaels, “β-Lactamase-producing Branhamella catarrhaliscausing otitis media in children,” J. Pediatr., 102:261-264, 1983.

Kyte & Doolittle, J. Mol. Biol., 157:105-132, 1982.

Leininger, Bowen, Renauld-Mongenie, Rouse, Menozzi, Locht, Heron,Brennan “Immunodominant domains present on the Bordetella pertussisvaccine component filamentous hemagglutinin,” J. Infect. Dis.,175:1423-1431, 1997.

Leininger, Bowen, Renauld-Mongenie, Rouse, Menozzi, Locht, Heron,Brennan

Maniatis, Fritsch and Sambrook, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1982.

Marchant, Am. J. Med., 88(Suppl. 5A):15S-19S, 1990.

McGehee, Am. J. Respir. Cell Mol. Biol., 1:201-210, 1989.

McLeod, Ahmad, Capewell, Croughan, Calder, Seaton, “Increase inbronchopulmonar infection due to Branhamella catarrhalis,” Br. Med. J.[Clin. Res]., 292:1103-1105, 1986.

Melendez & Johnson, Rev. Infect. Dis., 13:428-429, 1990.

Murphy and Bartos, “Surface exposed and antigenically conserveddeterminants of outer membrane proteins of Branhamella catarrhalis,”Infect. Immun., 57:2938-2941, 1989.

Murphy and Loeb, “lsolation of the outer membrane of Branhamellacatarrhalis,” Microb. Pathog., 6:159-174, 1989.

Murphy et al., Am. Jrnl. Med., 88:5A-41S-5A-45S, 1990.

Murphy, Kirkham and Lesse, “The major heat-modifiable outer membraneprotein CD is highly conserved among strains of Branhamellacatarrhalis,” Mol. Microbiol., 10:87-97, 1993.

Murphy, Pediat. Infect. Dis. J., 8: S75-S77, 1989.

Nicolas and Rubenstein, In: Vectors: A survey of molecular cloningvectors and their uses, Rodriguez and Denhardt (eds.), Stoneham:Butterworth, pp. 494-513, 1988.

Nicotra, Rivera, Liman, Wallace, “Branhamella catarrhalis as a lowerrespiratory tra(t pathogen in patients with chronic lung disease,” Arch.Intern. Med., 146:890-893, 1986.

Patrick, Kimura, Jackson, Hermanstorfer, Hood, McCracken Jr., Hansen,“Antigenic characterization of the oligosaccharide portion of thelipooligosaccharide of nontypable Haemophilus influenzae,” Infect.Immun., 55:2902-2911, 1987.

Pilz, Vocke, Heesemann, Brade, “Mechanism of YadA-mediated serumresistance of Yersinia enterocolitica serotype O3.” Infect. Immun.,60:189-195, 1992.

Reddy, Murphy, Faden, Bernstein, “Middle ear mucin glycoprotein;Purification and interaction with nontypeable Haemophilus influenzae andMoraxella catarrhalis,” Otolaryngol. Head Neck Surg., 116:175-180, 1997.

Ridgeway, In: Vectors: A survey of molecular cloning vectors and theiruses. Stoneham: Butterworth, Rodriguez R L, Denhardt D T, ed., pp.467-492, 1988.

Sambrook, Fritsch and Maniatis, Molecular Cloning—A Laboratory Manual,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

Sarubbi et al., Am. J. Med., 88(Suppl. 5A):9S-14S, 1990.

Schonheyder & Ejlertsen, Eur. J. Clin. Microbiol. Infect. Dis.,8:299-300, 1989.

Shine and Dalgarno, “Determinant of cistron specificity in bacterialribosomes,” Nature 254:34-38, 1975.

Skurnik and Wolf-Watz, “Analysis of the yopA gene encoding the Yop1virulence determinants of Yersinia spp.,” Mol. Microbiol., 3:517-529,1989.

Soto-Hernandez, Holtsclaw-Berk, Harvill, Berk, “Phenotypiccharacteristics of Branhamella catarrhalis strains,” J. Clin.Microbiol., 27:903-908, 1989.

St.Geme III and Falkow, “Haemophilus influenzae adheres to and enterscultured human epithelial cells,” Infect. Immun., 58:4036-4044, 1990.

St.Geme III, Cutter, Barenkamp, “Characterization of the genetic locusencoding Haemophilus influenzae type b surface fibrils,” J. Bacteriol.,178:6281-6287, 1996.

Stinchcomb et al., Nature, 282:39, 1979.

Temin, In: Gene Transfer, Kucherlapati (ed.), New York: Plenum Press,pp. 149-188, 1986.

Tissue Culture, Academic Press, Kruse and Patterson, editors, 1973.

Tschemper et al., Gene, 10:157, 1980.

Unhanand et al., J. Infect. Dis., 165:644-650, 1992.

Verduin, Bootsma, Hol, Fleer, Jansze, Klingman, Murphy, van Dijk,“Complement resistance in Moraxella (Branhamella) catarrhalis ismediated by a high-molecular-weight ouler membrane protein (HMW-OMP),”Abstracts General Meeting Amer. Soc. Microbiol., B137:189(Abstract),1995.

Verduin, Jansze, Hol, Mollnes, Verhoef, van Dijk, “Differences incomplement activation between complement-resistant andcomplement-sensitive Moraxella (Branhamella) catarrhalis strains occurat the level of membrane attack complex formation,” Infect. Immun.,62:589-595, 1994.

Verghese et al., J. Infect. Dis., 162:1189-92, 1990.

Weinberger et al., Science, 228:740-742, 1985.

Wolf et al., Comput. Appl. Biosci., 4(1):187-191, 1988.

Wright & Wallace, Semin. Respir. Infect., 4:40-46, 1989.

98 1 831 PRT Moraxella catarrhalis 1 Met Asn Lys Ile Tyr Lys Val Lys LysAsn Ala Ala Gly His Leu Val 1 5 10 15 Ala Cys Ser Glu Phe Ala Lys GlyHis Thr Lys Lys Ala Val Leu Gly 20 25 30 Ser Leu Leu Ile Val Gly Ala LeuGly Met Ala Thr Thr Ala Ser Ala 35 40 45 Gln Ala Thr Asn Ser Lys Gly ThrGly Ala His Ile Gly Val Asn Asn 50 55 60 Asn Asn Glu Ala Pro Gly Ser TyrSer Phe Ile Gly Ser Gly Gly Tyr 65 70 75 80 Asn Lys Ala Asp Arg Tyr SerAla Ile Gly Gly Gly Leu Phe Asn Lys 85 90 95 Ala Thr Asn Glu Tyr Ser ThrIle Val Gly Gly Gly Tyr Asn Lys Ala 100 105 110 Glu Gly Arg Tyr Ser ThrIle Gly Gly Gly Ser Asn Asn Glu Ala Thr 115 120 125 Asn Glu Tyr Ser ThrIle Val Gly Gly Asp Asp Asn Lys Ala Thr Gly 130 135 140 Arg Tyr Ser ThrIle Gly Gly Gly Asp Asn Asn Thr Arg Glu Gly Glu 145 150 155 160 Tyr SerThr Val Ala Gly Gly Lys Asn Asn Gln Ala Thr Gly Thr Gly 165 170 175 SerPhe Ala Ala Gly Val Glu Asn Gln Ala Asn Ala Glu Asn Ala Val 180 185 190Ala Val Gly Lys Lys Asn Ile Ile Glu Gly Glu Asn Ser Val Ala Ile 195 200205 Gly Ser Glu Asn Thr Val Lys Thr Glu His Lys Asn Val Phe Ile Leu 210215 220 Gly Ser Gly Thr Thr Gly Val Thr Ser Asn Ser Val Leu Leu Gly Asn225 230 235 240 Glu Thr Ala Gly Lys Gln Ala Thr Thr Val Lys Asn Ala GluVal Gly 245 250 255 Gly Leu Ser Leu Thr Gly Phe Ala Gly Glu Ser Lys AlaGlu Asn Gly 260 265 270 Val Val Ser Val Gly Ser Glu Gly Gly Glu Arg GlnIle Val Asn Val 275 280 285 Gly Ala Gly Gln Ile Ser Asp Thr Ser Thr AspAla Val Asn Gly Ser 290 295 300 Gln Leu His Ala Leu Ala Thr Val Val AspAsp Asn Gln Tyr Asp Ile 305 310 315 320 Val Asn Asn Arg Ala Asp Ile LeuAsn Asn Gln Asp Asp Ile Lys Asp 325 330 335 Leu Gln Lys Glu Val Lys GlyLeu Asp Asn Glu Val Gly Glu Leu Ser 340 345 350 Arg Asp Ile Asn Ser LeuHis Asp Val Thr Asp Asn Gln Gln Asp Asp 355 360 365 Ile Lys Glu Leu LysArg Gly Val Lys Glu Leu Asp Asn Glu Val Gly 370 375 380 Val Leu Ser ArgAsp Ile Asn Ser Leu His Asp Asp Val Ala Asp Asn 385 390 395 400 Gln AspAsp Ile Ala Lys Asn Lys Ala Asp Ile Lys Gly Leu Asn Lys 405 410 415 GluVal Lys Glu Leu Asp Lys Glu Val Gly Val Leu Ser Arg Asp Ile 420 425 430Gly Ser Leu His Asp Asp Val Ala Thr Asn Gln Ala Asp Ile Ala Lys 435 440445 Asn Gln Ala Asp Ile Lys Thr Leu Glu Asn Asn Val Glu Glu Glu Leu 450455 460 Leu Asn Leu Ser Gly Arg Leu Leu Asp Gln Lys Ala Asp Ile Asp Asn465 470 475 480 Asn Ile Asn Asn Ile Tyr Glu Leu Ala Gln Gln Gln Asp GlnHis Ser 485 490 495 Ser Asp Ile Lys Thr Leu Lys Asn Asn Val Glu Glu GlyLeu Leu Asp 500 505 510 Leu Ser Gly Arg Leu Ile Asp Gln Lys Ala Asp IleAla Lys Asn Gln 515 520 525 Ala Asp Ile Ala Gln Asn Gln Thr Asp Ile GlnAsp Leu Ala Ala Tyr 530 535 540 Asn Glu Leu Gln Asp Gln Tyr Ala Gln LysGln Thr Glu Ala Ile Asp 545 550 555 560 Ala Leu Asn Lys Ala Ser Ser GluAsn Thr Gln Asn Ile Ala Lys Asn 565 570 575 Gln Ala Asp Ile Ala Asn AsnIle Asn Asn Ile Tyr Glu Leu Ala Gln 580 585 590 Gln Gln Asp Gln His SerSer Asp Ile Lys Thr Leu Ala Lys Val Ser 595 600 605 Ala Ala Asn Thr AspArg Ile Ala Lys Asn Lys Ala Glu Ala Asp Ala 610 615 620 Ser Phe Glu ThrLeu Thr Lys Asn Gln Asn Thr Leu Ile Glu Gln Gly 625 630 635 640 Glu AlaLeu Val Glu Gln Asn Lys Ala Ile Asn Gln Glu Leu Glu Gly 645 650 655 PheAla Ala His Ala Asp Ile Gln Asp Lys Gln Ile Leu Gln Asn Gln 660 665 670Ala Asp Ile Thr Thr Asn Lys Thr Ala Ile Glu Gln Asn Ile Asn Arg 675 680685 Thr Val Ala Asn Gly Phe Glu Ile Glu Lys Asn Lys Ala Gly Ile Ala 690695 700 Thr Asn Lys Gln Glu Leu Ile Leu Gln Asn Asp Arg Leu Asn Arg Ile705 710 715 720 Asn Glu Thr Asn Asn Arg Gln Asp Gln Lys Ile Asp Gln LeuGly Tyr 725 730 735 Ala Leu Lys Glu Gln Gly Gln His Phe Asn Asn Arg IleSer Ala Val 740 745 750 Glu Arg Gln Thr Ala Gly Gly Ile Ala Asn Ala IleAla Ile Ala Thr 755 760 765 Leu Pro Ser Pro Ser Arg Ala Gly Glu His HisVal Leu Phe Gly Ser 770 775 780 Gly Tyr His Asn Gly Gln Ala Ala Val SerLeu Gly Ala Ala Gly Leu 785 790 795 800 Ser Asp Thr Gly Lys Ser Thr TyrLys Ile Gly Leu Ser Trp Ser Asp 805 810 815 Ala Gly Gly Leu Ser Gly GlyVal Gly Gly Ser Tyr Arg Trp Lys 820 825 830 2 3349 DNA Moraxellacatarrhalis 2 atcagcatgt gagcaaatga ctggcgtaaa tgactgatga gtgtctatttaatgaaagat 60 atcaatatat aaaagttgac tatagcgatg caatacagta aaatttgttacggctaaaca 120 taacgacggt ccaagatggc ggatatcgcc atttaccaac ctgataatcagtttgatagc 180 cattagcgat ggcatcaagt tgtgttgttg tattgtcata taaacggtaaatttggtttg 240 gtggatgccc catctgattt accgtccccc taataagtga gggggggggggagaccccag 300 tcatttatta ggagactaag atgaataaaa tttataaagt gaagaaaaatgccgcaggtc 360 acttggtggc atgttctgaa tttgccaaag gtcataccaa aaaggcagttttgggcagtt 420 tattgattgt tggggcgttg ggcatggcaa cgacggcgtc tgcacaagcaaccaacagca 480 aaggcacagg cgcgcacatc ggtgttaaca ataacaacga agccccaggcagttactctt 540 tcatcggtag tggcggttat aacaaagccg acagatactc tgccatcggtggtggccttt 600 ttaacaaagc cacaaacgag tactctacca tcgttggtgg cggttataacaaagccgaag 660 gcagatactc taccatcggt ggtggcagta acaacgaagc cacaaacgagtactctacca 720 tcgttggtgg cgatgacaac aaagccacag gcagatactc taccatcggtggtggcgata 780 acaacacacg cgaaggcgaa tactcaaccg tcgcaggggg caagaataaccaagccacag 840 gtacaggttc atttgccgca ggtgtagaga accaagccaa tgccgaaaacgccgtcgccg 900 tgggtaaaaa gaacattatc gaaggtgaaa actcagtagc catcggctctgagaataccg 960 ttaaaacaga acacaaaaat gtctttattc ttggctctgg cacaacaggtgtaacgagta 1020 actcagtgct actgggtaat gagaccgctg gcaaacaggc gaccactgttaagaatgccg 1080 aagtgggtgg tctaagccta acaggatttg caggggagtc aaaagctgaaaacggcgtag 1140 tttctgtggg tagtgaaggc ggtgagcgtc aaatcgttaa tgttggtgcaggtcagatca 1200 gtgacacctc aacagatgct gttaatggct cacagctaca tgctttggccacagttgttg 1260 atgacaacca atatgacatt gttaacaacc gagctgacat tcttaacaaccaagatgata 1320 tcaaagatct tcagaaggag gtgaaaggtc ttgataatga ggtgggtgaattaagccgag 1380 acattaattc acttcatgat gttactgaca accaacaaga tgacatcaaagagcttaaga 1440 ggggggtaaa agagcttgat aatgaggtgg gtgtattaag ccgagacattaattcacttc 1500 atgatgatgt tgctgacaac caagatgaca ttgctaaaaa caaagctgacatcaaaggtc 1560 ttaataagga ggtgaaagag cttgataagg aggtgggtgt attaagccgagacattggtt 1620 cacttcatga tgatgttgcc accaaccaag ctgacattgc taaaaaccaagcggatatca 1680 aaacacttga aaacaatgtc gaagaagaat tattaaatct aagcggtcgcctgcttgatc 1740 agaaagcgga tattgataat aacatcaaca atatctatga gctggcacaacagcaagatc 1800 agcatagctc tgatatcaaa acacttaaaa acaatgtcga agaaggtttattggatctaa 1860 gcggtcgcct cattgatcaa aaagcagata ttgctaaaaa ccaagctgacattgctcaaa 1920 accaaacaga catccaagat ctggccgctt acaatgagct acaagaccagtatgctcaaa 1980 agcaaaccga agcgattgac gctctaaata aagcaagctc tgagaatacacaaaacattg 2040 ctaaaaacca agcggatatt gctaataaca tcaacaatat ctatgagctggcacaacagc 2100 aagatcagca tagctctgat atcaaaacct tggcaaaagt aagtgctgccaatactgatc 2160 gtattgctaa aaacaaagct gaagctgatg caagttttga aacgctcaccaaaaatcaaa 2220 atactttgat tgagcaaggt gaagcattgg ttgagcaaaa taaagccatcaatcaagagc 2280 ttgaagggtt tgcggctcat gcagatattc aagataagca aattttacaaaaccaagctg 2340 atatcactac caataagacc gctattgaac aaaatatcaa tagaactgttgccaatgggt 2400 ttgagattga gaaaaataaa gctggtattg ctaccaataa gcaagagcttattcttcaaa 2460 atgatcgatt aaatcgaatt aatgagacaa ataatcgtca ggatcagaagattgatcaat 2520 taggttatgc actaaaagag cagggtcagc attttaataa tcgtattagtgctgttgagc 2580 gtcaaacagc tggaggtatt gcaaatgcta tcgcaattgc aactttaccatcgcccagta 2640 gagcaggtga gcatcatgtc ttatttggtt caggttatca caatggtcaagctgcggtat 2700 cattgggcgc ggctgggtta agtgatacag gaaaatcaac ttataagattggtctaagct 2760 ggtcagatgc aggtggatta tctggtggtg ttggtggcag ttaccgctggaaataaagcc 2820 taaatttaac tgctgtgtca aaaaatatgg tctgtataaa cagaccatatttttatccaa 2880 aaaaattatc ttaactttta taaagtatta taagccaaag ctgtaataataagagatgtt 2940 gaaataagag atgttaaagc tgctagacaa tcggcttgcg acgataaaataagatacctg 3000 gaatggacag ccccaaaacc aatgctgaga tgataaaaat cgcctcaaaaaaatgacgca 3060 tcataacgat aaataaatcc atatcaaatc caaaatagcc aatttgtaccatgctaacca 3120 tggctttata ggcagcgatt cccggcatca tacaaatcaa gctaggtacaatcaaggctt 3180 taggtggcag gccatgacgc tgagcaaaat gtacacccaa aaagctacccgccatcgccc 3240 caaagaatgt tgccacaacc aaatgcacac caaaaattac catcacttgttttaaaccaa 3300 aaccaagtgg tgttaccatc atgcaatgca tgatgtattg ctttgtcaa3349 3 573 PRT Moraxella catarrhalis 3 Met Lys Leu Leu Pro Leu Lys IleAla Val Thr Ser Ala Met Ile Val 1 5 10 15 Gly Leu Gly Ala Thr Ser ThrVal Asn Ala Gln Val Val Glu Gln Phe 20 25 30 Phe Pro Asn Ile Phe Phe AsnGlu Asn His Asp Glu Leu Asp Asp Ala 35 40 45 Tyr His Asn Met Ile Leu GlyAsp Thr Ala Ile Val Ser Asn Ser Gln 50 55 60 Asp Asn Ser Thr Gln Leu LysPhe Tyr Ser Asn Asp Glu Asp Ser Val 65 70 75 80 Pro Asp Ser Leu Leu PheSer Lys Leu Leu His Glu Gln Gln Leu Asn 85 90 95 Gly Phe Lys Ala Gly AspThr Ile Ile Pro Leu Asp Lys Asp Gly Lys 100 105 110 Pro Val Tyr Thr LysAsp Thr Arg Thr Lys Asp Gly Lys Val Glu Thr 115 120 125 Val Tyr Ser ValThr Thr Lys Ile Ala Thr Gln Asp Asp Val Glu Gln 130 135 140 Ser Ala TyrSer Arg Gly Ile Gln Gly Asp Ile Asp Asp Leu Tyr Asp 145 150 155 160 IleAsn Arg Glu Val Asn Glu Tyr Leu Lys Ala Thr His Asp Tyr Asn 165 170 175Glu Arg Gln Thr Glu Ala Ile Asp Ala Leu Asn Lys Ala Ser Ser Ala 180 185190 Asn Thr Asp Arg Ile Asp Thr Ala Glu Glu Arg Ile Asp Lys Asn Glu 195200 205 Tyr Asp Ile Lys Ala Leu Glu Ser Asn Val Glu Glu Gly Leu Leu Glu210 215 220 Leu Ser Gly His Leu Ile Asp Gln Lys Ala Asp Leu Thr Lys AspIle 225 230 235 240 Lys Ala Leu Glu Ser Asn Val Glu Glu Gly Leu Leu GluLeu Ser Gly 245 250 255 His Leu Ile Asp Gln Lys Ala Asp Leu Thr Lys AspIle Lys Ala Leu 260 265 270 Glu Ser Asn Val Glu Glu Gly Leu Leu Asp LeuSer Gly Arg Leu Leu 275 280 285 Asp Gln Lys Ala Asp Ile Ala Lys Asn GlnAla Asp Ile Ala Gln Asn 290 295 300 Gln Thr Asp Ile Gln Asp Leu Ala AlaTyr Asn Glu Leu Gln Asp Ala 305 310 315 320 Tyr Ala Lys Gln Gln Thr GluAla Ile Asp Ala Leu Asn Lys Ala Ser 325 330 335 Ser Glu Asn Thr Gln AsnIle Ala Lys Asn Gln Ala Asp Ile Ala Asn 340 345 350 Asn Ile Asn Asn IleTyr Glu Leu Ala Gln Gln Gln Asp Gln His Ser 355 360 365 Ser Asp Ile LysThr Leu Ala Lys Ala Ser Ala Ala Asn Thr Asp Arg 370 375 380 Ile Ala LysAsn Lys Ala Asp Ala Asp Ala Ser Phe Glu Thr Leu Thr 385 390 395 400 LysAsn Gln Asn Thr Leu Ile Glu Lys Asp Lys Glu His Asp Lys Leu 405 410 415Ile Thr Ala Asn Lys Thr Ala Ile Asp Ala Asn Lys Ala Ser Ala Asp 420 425430 Thr Lys Phe Ala Ala Thr Ala Asp Ala Ile Thr Lys Asn Gly Asn Ala 435440 445 Ile Thr Lys Asn Ala Lys Ser Ile Thr Asp Leu Gly Thr Lys Val Asp450 455 460 Gly Phe Asp Gly Arg Val Thr Ala Leu Asp Thr Lys Val Asn AlaLeu 465 470 475 480 Asp Thr Lys Val Asn Ala Phe Asp Gly Arg Ile Thr AlaLeu Asp Ser 485 490 495 Lys Val Glu Asn Gly Met Ala Ala Gln Ala Ala LeuSer Gly Leu Phe 500 505 510 Gln Pro Tyr Ser Val Gly Lys Phe Asn Ala ThrAla Ala Leu Gly Gly 515 520 525 Tyr Gly Ser Lys Ser Ala Val Ala Ile GlyAla Gly Tyr Arg Val Asn 530 535 540 Pro Asn Leu Ala Phe Lys Ala Gly AlaAla Ile Asn Thr Ser Gly Asn 545 550 555 560 Lys Lys Gly Ser Tyr Asn IleGly Val Asn Tyr Glu Phe 565 570 4 2596 DNA Moraxella catarrhalis 4ctggtggtcg cagggggcgt ctctgccaat cagtacacta cgccgcaccc tgaccgaaac 60gctccgccaa atcgatgcgt cggtgtacca tgccccgacc gagctatgca cggataatgg 120tgcgatgatc gcctatgctg gcttttgtcg gctaatccgt ggacagtcgg atgacttggt 180ggttcgctgc attccccgat gggatatgac gacgcttggc gtatctgctc ataaatagcc 240acatcaatca taccaaccaa atcataccaa ccaaatcgta caaacggttg atacatgcca 300aaaataccat attgaaagta gggtttgggt attatttatg taacttatat ctaatttggt 360gttgatactt tgataaagcc ttgctatact gtaacctaaa tggatatgat agagattttt 420ccatttatgc cagcaaaaga gatagataga tagatagata gatagataga tagatagata 480gatagataga tagatagata aaactctgtc ttttatctgt ccgctgatgc tttctgcctg 540ccaccgatga tatcatttat ctgcttttta ggcatcagtt atttcaccgt gatgactgat 600gtgatgactt aactaccaaa agagagtgct aaatgaaaac catgaaactt ctccccctaa 660aaatcgctgt aaccagtgcc atgattgttg gcttgggtgc gacatctact gtgaatgcac 720aagtagtgga acagtttttt ccgaatatct tttttaatga aaaccatgat gaattagatg 780atgcatacca taatatgatc ttaggggata ctgcgattgt atctaattca caagataata 840gtactcaatt gaaattttat tctaatgatg aagattcagt tcctgacagc ctactcttta 900gtaaactact tcatgagcag caacttaatg gttttaaagc aggtgacaca atcattcctt 960tggataagga tggcaaacct gtttatacaa aggacacgag aacaaaggat ggtaaagtag 1020aaacagttta ttcggtcacc accaaaatcg ctacccaaga tgatgttgaa caaagtgcat 1080attcacgagg cattcaaggt gatatcgatg atctgtatga cattaaccgt gaagtcaatg 1140aatacttaaa agcaacacat gattataatg aaagacaaac tgaagcaatt gacgctctaa 1200acaaagcaag ctctgcgaat actgatcgta ttgatactgc tgaagagcgt atcgataaaa 1260acgaatatga cattaaagca cttgaaagca atgtcgaaga aggtttgttg gagctaagcg 1320gtcacctcat tgatcaaaaa gcagatctta caaaagacat caaagcactt gaaagcaatg 1380tcgaagaagg tttgttggag ctaagcggtc acctcattga tcaaaaagca gatcttacaa 1440aagacatcaa agcacttgaa agcaatgtcg aagaaggttt gttggatcta agcggtcgtc 1500tgcttgatca aaaagcagat atcgctaaaa accaagctga cattgctcaa aaccaaacag 1560acatccaaga tctagccgct tacaacgagc tacaagatgc ctatgccaaa cagcaaaccg 1620aagcgattga cgctctaaac aaagcaagct ctgagaatac acaaaacatt gctaaaaacc 1680aagcggatat tgctaataac atcaacaata tctatgagct ggcacaacag caagatcagc 1740atagctctga tatcaaaacc ttggcaaaag caagtgctgc caatactgat cgtattgcta 1800aaaacaaagc cgatgctgat gcaagttttg aaacgctcac caaaaatcaa aatactttga 1860ttgaaaaaga taaagagcat gacaaattaa ttactgcaaa caaaactgcg attgatgcca 1920ataaagcatc tgcggatacc aagtttgcag cgacagcaga cgccattacc aaaaatggaa 1980atgctatcac taaaaacgca aaatctatca ctgatttggg tactaaagtg gatggttttg 2040acggtcgtgt aactgcatta gacaccaaag tcaatgcctt agacaccaaa gtcaatgcct 2100ttgatggtcg tatcacagct ttagacagta aagttgaaaa cggtatggct gcccaagctg 2160ccctaagtgg tctattccag ccttatagcg ttggtaagtt taatgcgacc gctgcacttg 2220gtggctatgg ctcaaaatct gcggttgcta tcggtgctgg ctatcgtgtg aatccaaatc 2280tggcgtttaa agctggtgcg gcgattaata ccagtggtaa taaaaaaggc tcttataaca 2340tcggtgtgaa ttacgagttt taattgtcta tcatcaccaa aaaaaagcag tcagtttact 2400ggctgctttt ttatgggttt ttgtggcttt tggttgtgag tgatggataa aagcttatca 2460agcgattgat gaatatcaat aaatgattgg taaatatcaa taaagcggtt tagggttttt 2520ggatatcttt taataagttt aaaaacccct gcataaaata aagctgggca tcagagctgc 2580gagtagcggc atacag 2596 5 892 PRT Moraxella catarrhalis 5 Met Asn Lys IleTyr Lys Val Lys Lys Asn Ala Ala Gly His Leu Val 1 5 10 15 Ala Cys SerGlu Phe Ala Lys Gly His Thr Lys Lys Ala Val Leu Gly 20 25 30 Ser Leu LeuIle Val Gly Ala Leu Gly Met Ala Thr Thr Ala Ser Ala 35 40 45 Gln Ala ThrLys Gly Thr Gly Lys His Val Val Asp Asn Lys Asp Asn 50 55 60 Lys Ala LysGly Asp Tyr Ser Thr Ala Ser Gly Gly Lys Asp Asn Glu 65 70 75 80 Ala LysGly Asn Tyr Ser Thr Val Gly Gly Gly Asp Tyr Asn Glu Ala 85 90 95 Lys GlyAsn Tyr Ser Thr Val Gly Gly Gly Ser Ser Asn Thr Ala Lys 100 105 110 GlyGlu Lys Ser Thr Ile Gly Gly Gly Asp Thr Asn Asp Ala Asn Gly 115 120 125Thr Tyr Ser Thr Ile Gly Gly Gly Tyr Tyr Ser Arg Ala Ile Gly Asp 130 135140 Ser Ser Thr Ile Gly Gly Gly Tyr Tyr Asn Gln Ala Thr Gly Glu Lys 145150 155 160 Ser Thr Val Ala Gly Gly Arg Asn Asn Gln Ala Thr Gly Asn AsnSer 165 170 175 Thr Val Ala Gly Gly Ser Tyr Asn Gln Ala Thr Gly Asn AsnSer Thr 180 185 190 Val Ala Gly Gly Ser His Asn Gln Ala Thr Gly Glu GlySer Phe Ala 195 200 205 Ala Gly Val Glu Asn Lys Ala Asn Ala Asn Asn AlaVal Ala Leu Gly 210 215 220 Lys Asn Asn Thr Ile Asp Gly Asp Asn Ser ValAla Ile Gly Ser Asn 225 230 235 240 Asn Thr Ile Asp Ser Gly Lys Gln AsnVal Phe Ile Leu Gly Ser Ser 245 250 255 Thr Asn Thr Thr Asn Ala Gln SerGly Ser Val Leu Leu Gly His Asn 260 265 270 Thr Ala Gly Lys Lys Ala ThrAla Val Ser Ser Ala Lys Val Asn Gly 275 280 285 Leu Thr Leu Gly Asn PheAla Gly Ala Ser Lys Thr Gly Asn Gly Thr 290 295 300 Val Ser Val Gly SerGlu Asn Asn Glu Arg Gln Ile Val Asn Val Gly 305 310 315 320 Ala Gly AsnIle Ser Ala Asp Ser Thr Asp Ala Val Asn Gly Ser Gln 325 330 335 Leu TyrAla Leu Ala Thr Ala Val Lys Ala Asp Ala Asp Glu Asn Phe 340 345 350 LysAla Leu Thr Lys Thr Gln Asn Thr Leu Ile Glu Gln Gly Glu Ala 355 360 365Gln Asp Ala Leu Ile Ala Gln Asn Gln Thr Asp Ile Thr Ala Asn Lys 370 375380 Thr Ala Ile Glu Arg Asn Phe Asn Arg Thr Val Val Asn Gly Phe Glu 385390 395 400 Ile Glu Lys Asn Lys Ala Gly Ile Ala Lys Asn Gln Ala Asp IleGln 405 410 415 Thr Leu Glu Asn Asn Val Gly Glu Glu Leu Leu Asn Leu SerGly Arg 420 425 430 Leu Leu Asp Gln Lys Ala Asp Ile Asp Asn Asn Ile AsnAsn Ile Tyr 435 440 445 Asp Leu Ala Gln Gln Gln Asp Gln His Ser Ser AspIle Lys Thr Leu 450 455 460 Lys Lys Asn Val Glu Glu Gly Leu Leu Asp LeuSer Gly Arg Leu Ile 465 470 475 480 Asp Gln Lys Ala Asp Leu Thr Lys AspIle Lys Thr Leu Glu Asn Asn 485 490 495 Val Glu Glu Gly Leu Leu Asp LeuSer Gly Arg Leu Ile Asp Gln Lys 500 505 510 Ala Asp Ile Ala Lys Asn GlnAla Asp Ile Ala Gln Asn Gln Thr Asp 515 520 525 Ile Gln Asp Leu Ala AlaTyr Asn Glu Leu Gln Asp Gln Tyr Ala Gln 530 535 540 Lys Gln Thr Glu AlaIle Asp Ala Leu Asn Lys Ala Ser Ser Ala Asn 545 550 555 560 Thr Asp ArgIle Ala Thr Ala Glu Leu Gly Ile Ala Glu Asn Lys Lys 565 570 575 Asp AlaGln Ile Ala Lys Ala Gln Ala Asn Glu Asn Lys Asp Gly Ile 580 585 590 AlaLys Asn Gln Ala Asp Ile Gln Leu His Asp Lys Lys Ile Thr Asn 595 600 605Leu Gly Ile Leu His Ser Met Val Ala Arg Ala Val Gly Asn Asn Thr 610 615620 Gln Gly Val Ala Thr Asn Lys Ala Asp Ile Ala Lys Asn Gln Ala Asp 625630 635 640 Ile Ala Asn Asn Ile Lys Asn Ile Tyr Glu Leu Ala Gln Gln GlnAsp 645 650 655 Gln His Ser Ser Asp Ile Lys Thr Leu Ala Lys Val Ser AlaAla Asn 660 665 670 Thr Asp Arg Ile Ala Lys Asn Lys Ala Glu Ala Asp AlaSer Phe Glu 675 680 685 Thr Leu Thr Lys Asn Gln Asn Thr Leu Ile Glu GlnGly Glu Ala Leu 690 695 700 Val Glu Gln Asn Lys Ala Ile Asn Gln Glu LeuGlu Gly Phe Ala Ala 705 710 715 720 His Ala Asp Val Gln Asp Lys Gln IleLeu Gln Asn Gln Ala Asp Ile 725 730 735 Thr Thr Asn Lys Ala Ala Ile GluGln Asn Ile Asn Arg Thr Val Ala 740 745 750 Asn Gly Phe Glu Ile Glu LysAsn Lys Ala Gly Ile Ala Thr Asn Lys 755 760 765 Gln Glu Leu Ile Leu GlnAsn Asp Arg Leu Asn Gln Ile Asn Glu Thr 770 775 780 Asn Asn Arg Gln AspGln Lys Ile Asp Gln Leu Gly Tyr Ala Leu Lys 785 790 795 800 Glu Gln GlyGln His Phe Asn Asn Arg Ile Ser Ala Val Glu Arg Gln 805 810 815 Thr AlaGly Gly Ile Ala Asn Ala Ile Ala Ile Ala Thr Leu Pro Ser 820 825 830 ProSer Arg Ala Gly Glu His His Val Leu Phe Gly Ser Gly Tyr His 835 840 845Asn Gly Gln Ala Ala Val Ser Leu Gly Ala Ala Gly Leu Ser Asp Thr 850 855860 Gly Lys Ser Thr Tyr Lys Ile Gly Leu Ser Trp Ser Asp Ala Gly Gly 865870 875 880 Leu Ser Gly Gly Val Gly Gly Ser Tyr Arg Trp Lys 885 890 63381 DNA Moraxella catarrhalis 6 tgtgagcaaa tgactggcgt aaatgactgatgaatgtcta tttaatgaaa gatatcaata 60 tataaaagtt gactatagcg atgcaatacagtaaaatttg ttacggctaa acataacgac 120 ggtccaagat ggcggatatc gccatttaccaacctgataa tcagtttgat agccattagc 180 gatggcatca agttgtgttg ttgtattgtcatataaacgg taaatttggt ttggtggatg 240 ccccatctga tttaccgtcc ccctaataagtgaggggggg ggagacccca gtcatttatt 300 aggagactaa gatgaacaaa atttataaagtgaaaaaaaa tgccgcaggt cacttggtgg 360 catgttctga atttgccaaa ggccataccaaaaaggcagt tttgggcagt ttattgattg 420 ttggggcatt gggcatggca acgacggcgtctgcacaagc aaccaaaggc acaggcaagc 480 acgttgttga caataaggac aacaaagccaaaggcgatta ctctaccgcc agtggtggca 540 aggacaacga agccaaaggc aattactctaccgtcggtgg tggcgattat aacgaagcca 600 aaggcaatta ctctaccgtc ggtggtggctctagtaatac cgccaaaggc gagaaatcaa 660 ccatcggtgg tggcgatact aacgacgccaacggcacata ctctaccatc ggtggtggct 720 attatagccg agccataggc gatagctctaccatcggtgg tggttattat aaccaagcca 780 caggcgagaa atcaacggtt gcagggggcaggaataacca agccacaggc aacaactcaa 840 cggttgcagg cggctcttat aaccaagccacaggcaacaa ctcaacggtt gcaggtggct 900 ctcataacca agccacaggt gaaggttcatttgcagcagg tgtagagaac aaagccaatg 960 ccaacaacgc cgtcgctcta ggtaaaaataacaccatcga tggcgataac tcagtagcca 1020 tcggctctaa taataccatt gacagtggcaaacaaaatgt ctttattctt ggctctagca 1080 caaacacaac aaatgcacaa agcggctccgtgctgctggg tcataatacc gctggcaaaa 1140 aagcaaccgc tgttagcagt gccaaagtgaacggcttaac cctaggaaat tttgcaggtg 1200 catcaaaaac tggtaatggt actgtatctgtcggtagtga gaataatgag cgtcaaatcg 1260 tcaatgttgg tgcaggtaat atcagtgctgattcaacaga tgctgttaat ggctcacagc 1320 tatatgcttt ggccacagct gtcaaagccgatgccgatga aaactttaaa gcactcacca 1380 aaactcaaaa tactttgatt gagcaaggtgaagcacaaga cgcattaatc gctcaaaatc 1440 aaactgacat cactgccaat aaaactgccattgagcgaaa ttttaataga actgttgtca 1500 atgggtttga gattgagaaa aataaagctggtattgctaa aaaccaagcg gatatccaaa 1560 cgcttgaaaa caatgtcgga gaagaactattaaatctaag cggtcgcctg cttgatcaaa 1620 aagcggatat tgataataac atcaacaatatctatgatct ggcacaacag caagatcagc 1680 atagctctga tatcaaaaca cttaaaaaaaatgtcgaaga aggtttgttg gatctaagtg 1740 gtcgcctcat tgatcaaaaa gcagatcttacgaaagacat caaaacactt gaaaacaatg 1800 tcgaagaagg tttgttggat ctaagcggtcgcctcattga tcaaaaagca gatattgcta 1860 aaaaccaagc tgacattgct caaaaccaaacagacatcca agatctggcc gcttacaacg 1920 agctacaaga ccagtatgct caaaagcaaaccgaagcgat tgacgctcta aataaagcaa 1980 gctctgccaa tactgatcgt attgctactgctgaattggg tatcgctgag aacaaaaaag 2040 acgctcagat cgccaaagca caagccaatgaaaataaaga cggcattgct aaaaaccaag 2100 ctgatatcca gttgcacgat aaaaaaatcaccaatctagg tatccttcac agcatggttg 2160 caagagcggt aggaaataac acacaaggtgttgctaccaa taaagctgac attgctaaaa 2220 accaagcaga tattgctaat aacatcaaaaatatctatga gctggcacaa cagcaagatc 2280 agcatagctc tgatatcaaa accttggcaaaagtaagtgc tgccaatact gatcgtattg 2340 ctaaaaacaa agctgaagct gatgcaagttttgaaacgct caccaaaaat caaaatactt 2400 tgattgagca aggtgaagca ttggttgagcaaaataaagc catcaatcaa gagcttgaag 2460 ggtttgcggc tcatgcagat gttcaagataagcaaatttt acaaaaccaa gctgatatca 2520 ctaccaataa ggccgctatt gaacaaaatatcaatagaac tgttgccaat gggtttgaga 2580 ttgagaaaaa taaagctggt attgctaccaataagcaaga gcttattctt caaaatgatc 2640 gattaaatca aattaatgag acaaataatcgtcaggatca gaagattgat caattaggtt 2700 atgcactaaa agagcagggt cagcattttaataatcgtat tagtgctgtt gagcgtcaaa 2760 cagctggagg tattgcaaat gctatcgcaattgcaacttt accatcgccc agtagagcag 2820 gtgagcatca tgtcttattt ggttcaggttatcacaatgg tcaagctgcg gtatcattgg 2880 gtgcggctgg gttaagtgat acaggaaaatcaacttataa gattggtcta agctggtcag 2940 atgcaggtgg attatctggt ggtgttggtggcagttaccg ctggaaatag agcctaaatt 3000 taactgctgt atcaaaaaat atggtctgtataaacagacc atatttttat ctaaaaactt 3060 atcttaactt ttatgaagca tcataagccaaagctgagta ataataagag atgttaaaat 3120 aagagatgtt aaaactgcta aacaatcggcttacgacgat aaaataaaat acctggaatg 3180 gacagcccca aaaccaatgc tgagatgataaaaatcgcct caaaaaaatg acgcatcata 3240 acgataaata aatccatatc aaatccaaaatagccaattt gtaccatgct aaccatggct 3300 ttataggcag cgattcccgg catcatacaaatcaagctag gtacaatcaa ggctttaggc 3360 ggcaggccat gacgctgagc a 3381 7 624PRT Moraxella catarrhalis 7 Val Asn Lys Ile Tyr Lys Val Lys Lys Asn AlaAla Gly His Ser Val 1 5 10 15 Ala Cys Ser Glu Phe Ala Lys Gly His ThrLys Lys Ala Val Leu Gly 20 25 30 Ser Leu Leu Ile Val Gly Ala Leu Gly MetAla Thr Thr Ala Ser Ala 35 40 45 Gln Thr Gly Ser Thr Asn Ala Ala Asn GlyAsn Ile Ile Ser Gly Val 50 55 60 Gly Ala Tyr Val Gly Gly Gly Val Ile AsnGln Ala Lys Gly Asn Tyr 65 70 75 80 Pro Thr Val Gly Gly Gly Phe Asp AsnArg Ala Thr Gly Asn Tyr Ser 85 90 95 Val Ile Ser Gly Gly Phe Asp Asn GlnAla Lys Gly Glu His Ser Thr 100 105 110 Ile Ala Gly Gly Glu Ser Asn GlnAla Thr Gly Arg Asn Ser Thr Val 115 120 125 Ala Gly Gly Ser Asn Asn GlnAla Val Gly Thr Asn Ser Thr Val Ala 130 135 140 Gly Gly Ser Asn Asn GlnAla Lys Gly Ala Asn Ser Phe Ala Ala Gly 145 150 155 160 Val Gly Asn GlnAla Asn Thr Asp Asn Ala Val Ala Leu Gly Lys Asn 165 170 175 Asn Thr IleAsn Gly Asn Asn Ser Ala Ala Ile Gly Ser Glu Asn Thr 180 185 190 Val AsnGlu Asn Gln Lys Asn Val Phe Ile Leu Gly Ser Asn Thr Thr 195 200 205 AsnAla Gln Ser Gly Ser Val Leu Leu Gly His Glu Thr Ser Gly Lys 210 215 220Glu Ala Thr Ala Val Ser Arg Ala Arg Val Asn Gly Leu Thr Leu Lys 225 230235 240 Asn Phe Ser Gly Val Ser Lys Ala Asp Asn Gly Thr Val Ser Val Gly245 250 255 Ser Gln Gly Lys Glu Arg Gln Ile Val His Val Gly Ala Gly GlnIle 260 265 270 Ser Asp Asp Ser Thr Asp Ala Val Asn Gly Ser Gln Leu TyrAla Leu 275 280 285 Ala Thr Ala Val Asp Asp Asn Gln Tyr Asp Ile Glu IleAsn Gln Asp 290 295 300 Asn Ile Lys Asp Leu Gln Lys Glu Val Lys Gly LeuAsp Lys Glu Val 305 310 315 320 Gly Val Leu Ser Arg Asp Ile Gly Ser LeuHis Asp Asp Val Ala Asp 325 330 335 Asn Gln Ala Asp Ile Ala Lys Asn LysAla Asp Ile Lys Glu Leu Asp 340 345 350 Lys Glu Met Asn Val Leu Ser ArgAsp Ile Val Ser Leu Asn Asp Asp 355 360 365 Val Ala Asp Asn Gln Ala AspIle Ala Lys Asn Gln Ala Asp Ile Lys 370 375 380 Thr Leu Glu Asn Asn ValGlu Glu Gly Leu Leu Asp Leu Ser Gly Arg 385 390 395 400 Leu Ile Asp GlnLys Ala Asp Ile Asp Asn Asn Ile Asn His Ile Tyr 405 410 415 Glu Leu AlaGln Gln Gln Asp Gln His Ser Ser Asp Ile Lys Thr Leu 420 425 430 Ala LysAla Ser Ala Ala Asn Thr Asp Arg Ile Ala Lys Asn Lys Ala 435 440 445 AspAla Asp Ala Ser Phe Glu Thr Leu Thr Lys Asn Gln Asn Thr Leu 450 455 460Ile Glu Lys Asp Lys Glu His Asp Lys Leu Ile Thr Ala Asn Lys Thr 465 470475 480 Ala Ile Asp Ala Asn Lys Ala Ser Ala Asp Thr Lys Phe Ala Ala Thr485 490 495 Ala Asp Ala Ile Thr Lys Asn Gly Asn Ala Ile Thr Lys Asn AlaLys 500 505 510 Ser Ile Thr Asp Leu Gly Thr Lys Val Asp Gly Phe Asp GlyArg Val 515 520 525 Thr Ala Leu Asp Thr Lys Val Asn Ala Phe Asp Gly ArgIle Thr Ala 530 535 540 Leu Asp Ser Lys Val Glu Asn Gly Met Ala Ala GlnAla Ala Leu Ser 545 550 555 560 Gly Leu Phe Gln Pro Tyr Ser Val Gly LysPhe Asn Ala Thr Ala Ala 565 570 575 Leu Gly Gly Tyr Gly Ser Lys Ser AlaVal Ala Ile Gly Ala Gly Tyr 580 585 590 Arg Val Asn Pro Asn Leu Ala PheLys Ala Gly Ala Ala Ile Asn Thr 595 600 605 Ser Gly Asn Lys Lys Gly SerTyr Asn Ile Gly Val Asn Tyr Glu Phe 610 615 620 8 3295 DNA Moraxellacatarrhalis 8 gccgcacctg accgagacgc tccgccaaat caatgcgtcg gtgtactatgccccgaccga 60 gctatgcacg gataatggtg cgatgatcgc ctatgctggc ttttgtcggctaagccgtgg 120 acagtcggat gacttggcgg ttcgctgcat tccccgatgg gatatgacaacgcttggtat 180 cgaatatgat aattaggctg tggtatttga gttttgagta atgtacctactaccactaat 240 ttatcataca atacataaac ataaaaaaca tcggtattgt taaaaaacaatacccaagtt 300 aaaatagctc aatactttac catagcacaa agaaacttgt gaacgaaacatttaataatt 360 gcccaaaatg tcactgcaca cactttgtaa aagcaggttt gggcaatggcaaacaacgat 420 acaaatgcaa aggttaccat cactattttt ctgtgaagca acgaagcaaccaaaaaagta 480 atgacattaa aaaaacaagc cattgataca aacagtaaac aaatcttaggctttgtctgt 540 ggtaaaacag acactaacac ctttaaacga ctttatcagc agttaaatacccataacatt 600 caactgtttt ttagtgacta ctggaaatct tatcgtcaag tcattttaaagccaaaacat 660 ataacaagca aagctcaaac ttttaccata gaggactata atagtctcattgggcatttc 720 atagcaagat ttacaagaaa gtcaaagtat tattctaaat ccgaaaaaatgatagaaaac 780 acgttgaatt tattatttgc taagtggaat ggtagcttaa gatatgtattttaatttaac 840 aatgccaaaa acatcaatta cagtaagatt ttaggcgttt tgcagttgctactttagtaa 900 agctttgtta tactagctgt taatatactc aagcttgttt gtgtttgagctatgtttatt 960 ttatagcagt agttggttat aaaatataaa taaagctaag ctcgagggtttggtaatggt 1020 tttttatgtt tataatacca acagagtatc tatacagcta aaatagctaataccttaggt 1080 gtattacaag taaaaatcct ttgttaatca gggagtgtat tatatgtatatttcctttgt 1140 atttggttat agcaatccct tggtaagaaa tcatatctat tttttattgttcaattattc 1200 aggagactaa ggtgaacaaa atttataaag tgaaaaaaaa tgccgcaggtcattcggtgg 1260 catgttctga atttgccaaa ggccatacca aaaaggcagt tttgggcagtttattgattg 1320 ttggggcatt gggcatggca acgacagcgt ctgcacaaac aggcagtacaaatgcagcca 1380 acggcaatat aatcagcggc gtaggcgcgt acgtcggtgg tggcgttataaaccaagcca 1440 aaggcaatta ccctaccgtc ggtggtggct ttgataaccg agccacaggcaattactctg 1500 tcatcagtgg tggctttgat aaccaagcca aaggcgagca ctctaccatcgcagggggtg 1560 agagtaacca agctacaggt cgtaactcaa cggttgcagg gggttctaataaccaagccg 1620 tgggtacaaa ctcaacggtt gcagggggtt ctaataacca agccaaaggtgcaaattcat 1680 ttgcagcagg tgtaggtaac caagccaata ccgacaacgc cgtcgctctaggtaaaaata 1740 acaccatcaa tggcaataac tcagcagcca tcggctctga gaataccgttaacgaaaatc 1800 aaaaaaatgt ctttattctt ggctctaaca caacaaatgc acaaagcggctcagtactgc 1860 taggtcatga aacctctggt aaagaagcga ccgctgttag cagagccagagtgaacggct 1920 taaccctaaa aaatttttca ggcgtatcaa aagctgataa tggtactgtatctgtcggta 1980 gtcagggtaa agagcgtcaa atcgttcatg ttggtgcagg tcagatcagtgatgattcaa 2040 cagatgctgt taatggctca cagctatatg ctttggctac agctgttgatgacaaccaat 2100 atgacattga aataaaccaa gataatatca aagatcttca gaaggaggtgaaaggtcttg 2160 ataaggaagt gggtgtatta agccgagaca ttggttcact tcatgatgatgttgctgaca 2220 accaagctga tattgctaaa aacaaagctg acatcaaaga gcttgataaggagatgaatg 2280 tattaagccg agacattgtc tcacttaatg atgatgttgc tgataaccaagctgacattg 2340 ctaaaaacca agcggatatc aaaacacttg aaaacaatgt cgaagaaggtttattggatc 2400 taagcggtcg cctcattgat caaaaagcag atattgataa taacatcaaccatatctatg 2460 agctggcaca acagcaagat cagcatagct ctgatatcaa aaccttggcaaaagcaagtg 2520 ctgccaatac tgatcgtatt gctaaaaaca aagccgatgc tgatgcaagttttgaaacac 2580 tcaccaaaaa tcaaaatact ttgattgaaa aagataaaga gcatgacaaattaattactg 2640 caaacaaaac tgcgattgat gccaataaag catctgcgga taccaagtttgcagcgacag 2700 cagacgccat taccaaaaat ggaaatgcta tcactaaaaa cgcaaaatctatcactgatt 2760 tgggtactaa agtggatggt tttgacggtc gtgtaactgc attagacaccaaagtcaatg 2820 cctttgatgg tcgcatcaca gctttagaca gtaaagttga aaacggtatggctgcccaag 2880 ctgccctaag tggtctattc cagccttata gcgttggtaa gtttaatgcgaccgctgcac 2940 ttggtggcta tggctcaaaa tctgcggttg ctatcggtgc tggctatcgtgtgaatccaa 3000 atctggcgtt taaagctggt gcggcgatta ataccagtgg caataaaaaaggctcttata 3060 acatcggtgt gaattacgag ttctaattgt ctatcatcac caaaaaaagcagtcagttta 3120 ctggctgctt ttttatgggt ttttatggct tttggttgtg agtgatggataaaagcttat 3180 caagcgattg atgaatatca ataaatgatt ggtaaatatc aataaagcggtttagggttt 3240 ttggatatct tttaataagt ttaaaaaccc ctgcataaaa taaagctggcatcag 3295 9 941 PRT Moraxella catarrhalis 9 Met Asn Lys Ile Tyr Lys ValLys Lys Asn Ala Ala Gly His Leu Val 1 5 10 15 Ala Cys Ser Glu Phe AlaLys Gly His Thr Lys Lys Ala Val Leu Gly 20 25 30 Ser Leu Leu Ile Val GlyIle Leu Gly Met Ala Thr Thr Ala Ser Ala 35 40 45 Gln Met Ala Thr Thr ProSer Ala Gln Val Val Lys Thr Asn Asn Lys 50 55 60 Lys Asn Gly Thr His ProPhe Ile Gly Gly Gly Asp Tyr Asn Thr Thr 65 70 75 80 Lys Gly Asn Tyr ProThr Ile Gly Gly Gly His Phe Asn Thr Ala Glu 85 90 95 Gly Asn Tyr Ser ThrVal Gly Gly Gly Phe Thr Asn Glu Ala Ile Gly 100 105 110 Lys Asn Ser ThrVal Gly Gly Gly Phe Thr Asn Glu Ala Met Gly Glu 115 120 125 Tyr Ser ThrVal Ala Gly Gly Ala Asn Asn Gln Ala Lys Gly Asn Tyr 130 135 140 Ser ThrVal Gly Gly Gly Asn Gly Asn Lys Ala Ile Gly Asn Asn Ser 145 150 155 160Thr Val Val Gly Gly Ser Asn Asn Gln Ala Lys Gly Glu His Ser Thr 165 170175 Ile Ala Gly Gly Lys Asn Asn Gln Ala Thr Gly Asn Gly Ser Phe Ala 180185 190 Ala Gly Val Glu Asn Lys Ala Asp Ala Asn Asn Ala Val Ala Leu Gly195 200 205 Asn Lys Asn Thr Ile Glu Gly Thr Asn Ser Val Ala Ile Gly SerAsn 210 215 220 Asn Thr Val Lys Thr Gly Lys Glu Asn Val Phe Ile Leu GlySer Asn 225 230 235 240 Thr Asn Thr Glu Asn Ala Gln Ser Gly Ser Val LeuLeu Gly Asn Asn 245 250 255 Thr Ala Gly Lys Ala Ala Thr Thr Val Asn AsnAla Glu Val Asn Gly 260 265 270 Leu Thr Leu Glu Asn Phe Ala Gly Ala SerLys Ala Asn Ala Asn Asn 275 280 285 Ile Gly Thr Val Ser Val Gly Ser GluAsn Asn Glu Arg Gln Ile Val 290 295 300 Asn Val Gly Ala Gly Gln Ile SerAla Thr Ser Thr Asp Ala Val Asn 305 310 315 320 Gly Ser Gln Leu His AlaLeu Ala Lys Ala Val Ala Lys Asn Lys Ser 325 330 335 Asp Ile Lys Gly LeuAsn Lys Gly Val Lys Glu Leu Asp Lys Glu Val 340 345 350 Gly Val Leu SerArg Asp Ile Asn Ser Leu His Asp Asp Val Ala Asp 355 360 365 Asn Gln AspSer Ile Ala Lys Asn Lys Ala Asp Ile Lys Gly Leu Asn 370 375 380 Lys GluVal Lys Glu Leu Asp Lys Glu Val Gly Val Leu Ser Arg Asp 385 390 395 400Ile Gly Ser Leu His Asp Asp Val Ala Asp Asn Gln Asp Ser Ile Ala 405 410415 Lys Asn Lys Ala Asp Ile Lys Gly Leu Asn Lys Glu Val Lys Glu Leu 420425 430 Asp Lys Glu Val Gly Val Leu Ser Arg Asp Ile Gly Ser Leu His Asp435 440 445 Asp Val Ala Thr Asn Gln Ala Asp Ile Ala Lys Asn Gln Ala AspIle 450 455 460 Lys Thr Leu Glu Asn Asn Val Glu Glu Glu Leu Leu Asn LeuSer Gly 465 470 475 480 Arg Leu Ile Asp Gln Lys Ala Asp Ile Asp Asn AsnIle Asn Asn Ile 485 490 495 Tyr Glu Leu Ala Gln Gln Gln Asp Gln His SerSer Asp Ile Lys Thr 500 505 510 Leu Lys Asn Asn Val Glu Glu Gly Leu LeuAsp Leu Ser Gly Arg Leu 515 520 525 Ile Asp Gln Lys Ala Asp Leu Thr LysAsp Ile Lys Thr Leu Lys Asn 530 535 540 Asn Val Glu Glu Gly Leu Leu AspLeu Ser Gly Arg Leu Ile Asp Gln 545 550 555 560 Lys Ala Asp Ile Ala LysAsn Gln Ala Asp Ile Ala Gln Asn Gln Thr 565 570 575 Asp Ile Gln Asp LeuAla Ala Tyr Asn Glu Leu Gln Asp Gln Tyr Ala 580 585 590 Gln Lys Gln ThrGlu Ala Ile Asp Ala Leu Asn Lys Ala Ser Ser Ala 595 600 605 Asn Thr AspArg Ile Ala Thr Ala Glu Leu Gly Ile Ala Glu Asn Lys 610 615 620 Lys AspAla Gln Ile Ala Lys Ala Gln Ala Asn Glu Asn Lys Asp Gly 625 630 635 640Ile Ala Lys Asn Gln Ala Asp Ile Gln Leu His Asp Lys Lys Ile Thr 645 650655 Asn Leu Gly Ile Leu His Ser Met Val Ala Arg Ala Val Gly Asn Asn 660665 670 Thr Gln Gly Val Ala Thr Asn Lys Ala Asp Ile Ala Lys Asn Gln Ala675 680 685 Asp Ile Ala Asn Asn Ile Lys Asn Ile Tyr Glu Leu Ala Gln GlnGln 690 695 700 Asp Gln His Ser Ser Asp Ile Lys Thr Leu Ala Lys Val SerAla Ala 705 710 715 720 Asn Thr Asp Arg Ile Ala Lys Asn Lys Ala Glu AlaAsp Ala Ser Phe 725 730 735 Glu Thr Leu Thr Lys Asn Gln Asn Thr Leu IleGlu Gln Gly Glu Ala 740 745 750 Leu Val Glu Gln Asn Lys Ala Ile Asn GlnGlu Leu Glu Gly Phe Ala 755 760 765 Ala His Ala Asp Val Gln Asp Lys GlnIle Leu Gln Asn Gln Ala Asp 770 775 780 Ile Thr Thr Asn Lys Thr Ala IleGlu Gln Asn Ile Asn Arg Thr Val 785 790 795 800 Ala Asn Gly Phe Glu IleGlu Lys Asn Lys Ala Gly Ile Ala Thr Asn 805 810 815 Lys Gln Glu Leu IleLeu Gln Asn Asp Arg Leu Asn Gln Ile Asn Glu 820 825 830 Thr Asn Asn HisGln Asp Gln Lys Ile Asp Gln Leu Gly Tyr Ala Leu 835 840 845 Lys Glu GlnGly Gln His Phe Asn Asn Arg Ile Ser Ala Val Glu Arg 850 855 860 Gln ThrAla Gly Gly Ile Ala Asn Ala Ile Ala Ile Ala Thr Leu Pro 865 870 875 880Ser Pro Ser Arg Ala Gly Glu His His Val Leu Phe Gly Ser Gly Tyr 885 890895 His Asn Gly Gln Ala Ala Val Ser Leu Gly Ala Ala Gly Leu Ser Asp 900905 910 Thr Gly Lys Ser Thr Tyr Lys Ile Gly Leu Ser Trp Ser Asp Ala Gly915 920 925 Gly Leu Ser Gly Gly Val Gly Gly Ser Tyr Arg Trp Lys 930 935940 10 3538 DNA Moraxella catarrhalis 10 ttctgtgagc aaatgactggcgtaaatgac tgatgagtgt ctatttaatg aaagatatca 60 atatataaaa gttgactatagcgatgcaat acagtaaaat ttgttacggc taaacataac 120 gacggtccaa gatggcggatatcgccattt accaacctga taatcagttt gatagccatt 180 agcgatggca tcaagttgtgttgttgtatt gtcatataaa cggtaaattt ggtttggtgg 240 atgccccatc tgatttaccgtccccctaat aagtgagggg gggggggaga ccccagtcat 300 ttattaggag actaagatgaacaaaattta taaagtgaaa aaaaatgccg caggtcactt 360 ggtggcgtgt tctgaatttgccaaaggtca taccaaaaag gcagttttgg gcagtttatt 420 gattgttgga atattgggtatggcaacgac agcatctgca caaatggcaa cgacgccgtc 480 tgcacaagta gtcaagacaaacaataaaaa aaacggcacg caccctttca tcggtggtgg 540 cgattataat accaccaaaggcaattaccc taccatcggt ggtggccatt ttaataccgc 600 cgaaggcaat tactctaccgtcggtggtgg ctttactaac gaagccatag gcaagaactc 660 taccgtcggt ggtggctttactaacgaagc catgggcgaa tactcaaccg tcgcaggcgg 720 tgctaacaac caagccaaaggcaattactc taccgtcggt ggtggcaatg gcaacaaagc 780 cataggcaac aactcaacggttgtaggtgg ttctaacaac caagccaaag gcgagcactc 840 taccatcgca gggggcaagaataaccaagc tacaggtaat ggttcatttg cagcaggtgt 900 agagaacaaa gccgatgctaacaacgccgt cgctctaggt aacaagaaca ccatcgaagg 960 tacaaactca gtagccatcggctctaataa taccgttaaa actggcaaag aaaatgtctt 1020 tattcttggc tctaacacaaacacagaaaa tgcacaaagt ggctccgtgc tgctgggtaa 1080 taataccgct ggcaaagcagcgaccactgt taacaatgcc gaagtgaacg gcttaaccct 1140 agaaaatttt gcaggtgcatcaaaagctaa tgctaataat attggtactg tatctgtcgg 1200 tagtgagaat aatgagcgtcaaatcgttaa tgttggtgca ggtcagatca gtgccacctc 1260 aacagatgct gttaatggctcacagctaca tgctttagcc aaagctgttg ctaaaaacaa 1320 atctgacatc aaaggtcttaataagggggt gaaagagctt gataaggagg tgggtgtatt 1380 aagccgagac attaattcacttcatgatga tgttgctgac aaccaagata gcattgctaa 1440 aaacaaagct gacatcaaaggtcttaataa ggaggtgaaa gagcttgata aggaggtggg 1500 tgtattaagc cgagacattggttcacttca tgatgatgtt gctgacaacc aagatagcat 1560 tgctaaaaac aaagctgacatcaaaggtct taataaggag gtgaaagagc ttgataagga 1620 ggtgggtgta ttaagccgagacattggttc acttcatgat gatgttgcca ccaaccaagc 1680 tgacattgct aaaaaccaagcggatatcaa aacacttgaa aacaatgtcg aagaagaatt 1740 attaaatcta agcggtcgcctcattgatca aaaagcggat attgataata acatcaacaa 1800 tatctatgag ctggcacaacagcaagatca gcatagctct gatatcaaaa cacttaaaaa 1860 caatgtcgaa gaaggtttgttggatctaag cggtcgcctc attgatcaaa aagcagatct 1920 tacgaaagac atcaaaacacttaaaaacaa tgtcgaagaa ggtttattgg atctaagcgg 1980 tcgcctcatt gatcaaaaagcagatattgc taaaaaccaa gctgacattg ctcaaaacca 2040 aacagacatc caagatctggccgcttacaa cgagctacaa gaccagtatg ctcaaaagca 2100 aaccgaagcg attgacgctctaaataaagc aagctctgcc aatactgatc gtattgctac 2160 tgctgaattg ggtatcgctgagaacaaaaa agacgctcag atcgccaaag cacaagccaa 2220 tgaaaataaa gacggcattgctaaaaacca agctgatatc cagttgcacg ataaaaaaat 2280 caccaatcta ggtatccttcacagcatggt tgcaagagcg gtaggaaata atacacaagg 2340 tgttgctacc aacaaagctgatattgctaa aaaccaagca gatattgcta ataacatcaa 2400 aaatatctat gagctggcacaacagcaaga tcagcatagc tctgatatca aaaccttggc 2460 aaaagtaagt gctgccaatactgatcgtat tgctaaaaac aaagctgaag ctgatgcaag 2520 ttttgaaacg ctcaccaaaaatcaaaatac tttgattgag caaggtgaag cattggttga 2580 gcaaaataaa gccatcaatcaagagcttga agggtttgcg gctcatgcag atgttcaaga 2640 taagcaaatt ttacaaaaccaagctgatat cactaccaat aagaccgcta ttgaacaaaa 2700 tatcaataga actgttgccaatgggtttga gattgagaaa aataaagctg gtattgctac 2760 caataagcaa gagcttattcttcaaaatga tcgattaaat caaattaatg agacaaataa 2820 tcatcaggat cagaagattgatcaattagg ttatgcacta aaagagcagg gtcagcattt 2880 taataatcgt attagtgctgttgagcgtca aacagctgga ggtattgcaa atgctatcgc 2940 aattgcaact ttaccatcgcccagtagagc aggtgagcat catgtcttat ttggttcagg 3000 ttatcacaat ggtcaagctgcggtatcatt gggcgcggct ggattaagtg atacaggaaa 3060 atcaacttat aagattggtctaagctggtc agatgcaggt ggattatctg gtggtgttgg 3120 tggcagttac cgctggaaatagagcctaaa tttaactgct gtatcaaaaa atatggtctg 3180 tataaacaga ccatatttttatctaaaaaa cttatcttaa cttttatgaa gcatcataag 3240 ccaaagctga gtaataataagagatgttaa aataagagat gttaaaactg ctaaacaatc 3300 ggcttgcgac gataaaataaaatacctgga atggacagcc ccaaaaccaa tgctgagatg 3360 ataaaaatcg cctcaaaaaaatgacgcatc ataacgataa ataaatccat atcaaatcca 3420 aaatagccaa tttgtaccatgctaaccatg gctttatagg cagcgattcc cggcatcata 3480 caaatcaagc taggtacaatcaaggcttta ggcggcaggc catgacgctg agcaaaaa 3538 11 610 PRT Moraxellacatarrhalis 11 Met Lys Leu Leu Pro Leu Lys Ile Ala Val Thr Ser Ala MetIle Ile 1 5 10 15 Gly Leu Gly Ala Ala Ser Thr Ala Asn Ala Gln Ser ArgAsp Arg Ser 20 25 30 Leu Glu Asp Ile Gln Asp Ser Ile Ser Lys Leu Val GlnAsp Asp Ile 35 40 45 Asp Thr Leu Lys Gln Asp Gln Gln Lys Met Asn Lys TyrLeu Leu Leu 50 55 60 Asn Gln Leu Ala Asn Thr Leu Ile Thr Asp Glu Leu AsnAsn Asn Val 65 70 75 80 Ile Lys Asn Thr Asn Ser Ile Glu Ala Leu Gly AspGlu Ile Gly Trp 85 90 95 Leu Glu Asn Asp Ile Ala Asp Leu Glu Glu Gly ValGlu Glu Leu Thr 100 105 110 Lys Asn Gln Asn Thr Leu Ile Glu Lys Asp GluGlu His Asp Arg Leu 115 120 125 Ile Ala Gln Asn Gln Ala Asp Ile Gln ThrLeu Glu Asn Asn Val Val 130 135 140 Glu Glu Leu Phe Asn Leu Ser Gly ArgLeu Ile Asp Gln Glu Ala Asp 145 150 155 160 Ile Ala Lys Asn Asn Ala SerIle Glu Glu Leu Tyr Asp Phe Asp Asn 165 170 175 Glu Val Ala Glu Arg IleGly Glu Ile His Ala Tyr Thr Glu Glu Val 180 185 190 Asn Lys Thr Leu GluAsn Leu Ile Thr Asn Ser Val Lys Asn Thr Asp 195 200 205 Asn Ile Asp LysAsn Lys Ala Asp Ile Asp Asn Asn Ile Asn His Ile 210 215 220 Tyr Glu LeuAla Gln Gln Gln Asp Gln His Ser Ser Asp Ile Lys Thr 225 230 235 240 LeuLys Asn Asn Val Glu Glu Gly Leu Leu Glu Leu Ser Gly His Leu 245 250 255Ile Asp Gln Lys Ala Asp Leu Thr Lys Asp Ile Lys Ala Leu Glu Ser 260 265270 Asn Val Glu Glu Gly Leu Leu Asp Leu Ser Gly Arg Leu Leu Asp Gln 275280 285 Lys Ala Asp Leu Thr Lys Asp Ile Lys Ala Leu Glu Ser Asn Val Glu290 295 300 Glu Gly Leu Leu Asp Leu Ser Gly Arg Leu Leu Asp Gln Lys AlaAsp 305 310 315 320 Ile Ala Gln Asn Gln Thr Asp Ile Gln Asp Leu Ala AlaTyr Asn Glu 325 330 335 Leu Gln Asp Gln Tyr Ala Gln Lys Gln Thr Glu AlaIle Asp Ala Leu 340 345 350 Asn Lys Ala Ser Ser Glu Asn Thr Gln Asn IleGlu Asp Leu Ala Ala 355 360 365 Tyr Asn Glu Leu Gln Asp Ala Tyr Ala LysGln Gln Thr Glu Ala Ile 370 375 380 Asp Ala Leu Asn Lys Ala Ser Ser GluAsn Thr Gln Asn Ile Ala Lys 385 390 395 400 Asn Gln Ala Asp Ile Ala AsnAsn Ile Asn Asn Ile Tyr Glu Leu Ala 405 410 415 Gln Gln Gln Asp Gln HisSer Ser Asp Ile Lys Thr Leu Ala Lys Ala 420 425 430 Ser Ala Ala Asn ThrAsn Arg Ile Ala Thr Ala Glu Leu Gly Ile Ala 435 440 445 Glu Asn Lys LysAsp Ala Gln Ile Ala Lys Ala Gln Ala Asn Ala Asn 450 455 460 Lys Thr AlaIle Asp Glu Asn Lys Ala Ser Ala Asp Thr Lys Phe Ala 465 470 475 480 AlaThr Ala Asp Ala Ile Thr Lys Asn Gly Asn Ala Ile Thr Lys Asn 485 490 495Ala Lys Ser Ile Thr Asp Leu Gly Thr Lys Val Asp Gly Phe Asp Gly 500 505510 Arg Val Thr Ala Leu Asp Thr Lys Val Asn Ala Phe Asp Gly Arg Ile 515520 525 Thr Ala Leu Asp Ser Lys Val Glu Asn Gly Met Ala Ala Gln Ala Ala530 535 540 Leu Ser Gly Leu Phe Gln Pro Tyr Ser Val Gly Lys Phe Asn AlaThr 545 550 555 560 Ala Ala Leu Gly Gly Tyr Gly Ser Lys Ser Ala Val AlaIle Gly Ala 565 570 575 Gly Tyr Arg Val Asn Pro Asn Leu Ala Phe Lys AlaGly Ala Ala Ile 580 585 590 Asn Thr Ser Gly Asn Lys Lys Gly Ser Tyr AsnIle Gly Val Asn Tyr 595 600 605 Glu Phe 610 12 2673 DNA Moraxellacatarrhalis 12 ccatcagtac atacgccgca cctgaccgag acgctccgcc aaatcaatgcgtcggtgtac 60 tacgccccga ccgagctatg cacggataat ggtgcgatga tcgcttacgctggcttttgt 120 cggctaagcc gtggacagtc ggatgacttg gcggttcgct gcattccccgatgggatatg 180 acaacgcttg gcgtatctgc tcatagatag ccacatcaat cataccaacgatattggtat 240 ataccaaatt gatacctgcc aaaaatacca tattgaaagt agggtttgggtattatttat 300 gtaacttata tctaatttgg tgttgatact ttgataaagc cttgctatactgtaacctaa 360 atggatatga tagagatttt tccatttatg ccagcaaaag agatagatagatagatagat 420 agatagatag atagatagat agatagatag atagataaaa ctctgtcttttatctgtcca 480 ctgatgcttt ctgcctgcca ccgatgatat cgtttatctg cttttttaggcatcagttat 540 ttcaccgtga tgactgatgt gatgacttaa ccaccaaaag agagtgctaaatgaaaacca 600 tgaaacttct ccctctaaaa atcgctgtaa ccagtgccat gattattggtttgggtgcgg 660 catctactgc gaatgcacag tctcgggata gatctttaga agatatacaagattcaatta 720 gtaaacttgt tcaagatgat atagatacac taaaacaaga tcagcagaagatgaacaagt 780 atctgttgct caaccagtta gctaatactt taattacaga cgagctcaacaataatgtta 840 taaaaaacac caattctatt gaagctcttg gtgatgagat tggatggcttgaaaatgata 900 ttgcagactt ggaagaaggt gttgaagaac tcaccaaaaa ccaaaatactttgattgaaa 960 aagatgaaga gcatgacaga ttaatcgctc aaaatcaagc tgatatccaaacacttgaaa 1020 acaatgtcgt agaagaacta ttcaatctaa gcggtcgcct aattgatcaagaagcggata 1080 ttgctaaaaa taatgcttct attgaagagc tttatgattt tgataatgaggttgcagaaa 1140 ggataggtga gatacatgct tatactgaag aggtaaataa aactcttgaaaacttgataa 1200 caaacagtgt taagaatact gataatattg acaaaaacaa agctgatattgataataaca 1260 tcaaccatat ctatgagctg gcacaacagc aagatcagca tagctctgatatcaaaacac 1320 ttaaaaacaa tgtcgaagaa ggtttgttgg agctaagcgg tcacctcattgatcaaaaag 1380 cggatcttac aaaagacatc aaagcacttg aaagcaatgt cgaagaaggtttgttggatc 1440 taagcggtcg tctgcttgat caaaaagcgg atcttacaaa agacatcaaagcacttgaaa 1500 gcaatgtcga agaaggtttg ttggatctaa gcggtcgtct gcttgatcaaaaagcggata 1560 ttgctcaaaa ccaaacagac atccaagatc tggccgctta caacgagctacaagaccagt 1620 atgctcaaaa gcaaaccgaa gcgattgacg ctctaaataa agcaagctctgagaatacac 1680 aaaacatcga agatctggcc gcttacaatg agctacaaga tgcctatgccaaacagcaaa 1740 ccgaagcgat tgacgctcta aataaagcaa gctctgagaa tacacaaaacattgctaaaa 1800 accaagcgga tattgctaat aacatcaaca atatctatga gctggcacaacagcaagatc 1860 agcatagctc tgatatcaaa accttggcaa aagcaagtgc tgccaatactaatcgtattg 1920 ctactgctga attgggcatc gctgagaaca aaaaagacgc tcagatcgccaaagcacaag 1980 cgaatgccaa caaaactgcg attgatgaaa acaaagcatc tgcggataccaagtttgcag 2040 caacagcaga cgccattacc aaaaatggaa atgctatcac taaaaacgcaaaatctatca 2100 ctgatttggg cactaaagtg gatggttttg acggtcgtgt aactgcattagacaccaaag 2160 tcaatgcctt tgatggtcgt atcacagctt tagacagtaa agttgaaaacggtatggctg 2220 cccaagctgc cctaagtggt ctattccagc cttatagcgt tggtaagtttaatgcgaccg 2280 ctgcacttgg tggctatggc tcaaaatctg cggttgctat cggtgctggctatcgtgtga 2340 atccaaatct ggcgtttaaa gctggtgcgg cgattaatac cagtggcaataaaaaaggct 2400 cttataacat cggtgtgaat tacgagttct aattgtctat catcaccaaaaaaagcagtc 2460 agtttactgg ctgctttttt atgggttttt gtggcttttg gttgtgagtgatggataaaa 2520 gcttatcaag cgattgatga atatcaataa atgattggta aatatcaataaagcggttta 2580 gggtttttgg atatctttta ataagtttaa aaacccctgc ataaaataaagctgggcatc 2640 agagctgcga gtagcggcat acagcgggag atc 2673 13 873 PRTMoraxella catarrhalis 13 Met Asn Lys Ile Tyr Lys Val Lys Lys Asn Ala AlaGly His Leu Val 1 5 10 15 Ala Cys Ser Glu Phe Ala Lys Gly His Thr LysLys Ala Val Leu Gly 20 25 30 Ser Leu Leu Ile Val Gly Ile Leu Gly Met AlaThr Thr Ala Ser Ala 35 40 45 Gln Gln Thr Ile Ala Arg Gln Gly Lys Gly MetHis Ser Ile Ile Gly 50 55 60 Gly Gly Asn Asp Asn Glu Ala Asn Gly Asp TyrSer Thr Val Ser Gly 65 70 75 80 Gly Asp Tyr Asn Glu Ala Lys Gly Asp SerSer Thr Ile Gly Gly Gly 85 90 95 Tyr Tyr Asn Glu Ala Asn Gly Asp Ser SerThr Ile Gly Gly Gly Phe 100 105 110 Tyr Asn Glu Ala Lys Gly Glu Ser SerThr Ile Gly Gly Gly Asp Asn 115 120 125 Asn Ser Ala Thr Gly Met Tyr SerThr Ile Gly Gly Gly Asp Asn Asn 130 135 140 Ser Ala Thr Gly Arg Tyr SerThr Ile Ala Gly Gly Trp Leu Asn Gln 145 150 155 160 Ala Thr Gly His SerSer Thr Val Ala Gly Gly Trp Leu Asn Gln Ala 165 170 175 Thr Asn Glu AsnSer Thr Val Gly Gly Gly Arg Phe Asn Gln Ala Thr 180 185 190 Gly Arg AsnSer Thr Val Ala Gly Gly Tyr Lys Asn Lys Ala Thr Gly 195 200 205 Val AspSer Thr Ile Ala Gly Gly Arg Asn Asn Gln Ala Asn Gly Ile 210 215 220 GlySer Phe Ala Ala Gly Ile Asp Asn Gln Ala Asn Ala Asn Asn Thr 225 230 235240 Val Ala Leu Gly Asn Lys Asn Ile Ile Lys Gly Lys Asp Ser Val Ala 245250 255 Ile Gly Ser Asn Asn Thr Val Glu Thr Gly Lys Glu Asn Val Phe Ile260 265 270 Leu Gly Ser Asn Thr Lys Asp Ala His Ser Asn Ser Val Leu LeuGly 275 280 285 Asn Glu Thr Thr Gly Lys Ala Ala Thr Thr Val Glu Asn AlaLys Val 290 295 300 Gly Gly Leu Ser Leu Thr Gly Phe Val Gly Ala Ser LysAla Asn Thr 305 310 315 320 Asn Asn Gly Thr Val Ser Val Gly Lys Gln GlyLys Glu Arg Gln Ile 325 330 335 Val Asn Val Gly Ala Gly Gln Ile Arg AlaAsp Ser Thr Asp Ala Val 340 345 350 Asn Gly Ser Gln Leu His Ala Leu AlaThr Ala Val Asp Ala Glu Phe 355 360 365 Arg Thr Leu Thr Gln Thr Gln AsnAla Leu Ile Glu Gln Gly Glu Ala 370 375 380 Ile Asn Gln Glu Leu Glu GlyLeu Ala Asp Tyr Thr Asn Ala Gln Asp 385 390 395 400 Glu Lys Ile Leu LysAsn Gln Thr Asp Ile Thr Ala Asn Lys Thr Ala 405 410 415 Ile Glu Gln AsnPhe Asn Arg Thr Val Thr Asn Gly Phe Glu Ile Glu 420 425 430 Lys Asn LysAla Gly Ile Ala Lys Asn Gln Ala Asp Ile Gln Thr Leu 435 440 445 Glu AsnAsp Val Gly Lys Glu Leu Leu Asn Leu Ser Gly Arg Leu Leu 450 455 460 AspGln Lys Ala Asp Ile Asp Asn Asn Ile Asn Asn Ile Tyr Glu Leu 465 470 475480 Ala Gln Gln Gln Asp Gln His Ser Ser Asp Ile Lys Thr Leu Lys Asn 485490 495 Asn Val Glu Glu Gly Leu Leu Asp Leu Ser Gly Arg Leu Ile Asp Gln500 505 510 Lys Ala Asp Leu Thr Lys Asp Ile Lys Ala Leu Glu Asn Asn ValGlu 515 520 525 Glu Gly Leu Leu Asp Leu Ser Gly Arg Leu Ile Asp Gln LysAla Asp 530 535 540 Ile Ala Lys Asn Gln Ala Asp Ile Gln Asp Leu Ala AlaTyr Asn Glu 545 550 555 560 Leu Gln Asp Gln Tyr Ala Gln Lys Gln Thr GluAla Ile Asp Ala Leu 565 570 575 Asn Lys Ala Ser Ser Ala Asn Thr Asp ArgIle Ala Thr Ala Glu Leu 580 585 590 Gly Ile Ala Glu Asn Lys Lys Asp AlaGln Ile Ala Lys Ala Gln Ala 595 600 605 Asn Glu Asn Lys Asp Gly Ile AlaLys Asn Gln Ala Asp Ile Ala Asn 610 615 620 Asn Ile Lys Asn Ile Tyr GluLeu Ala Gln Gln Gln Asp Gln His Ser 625 630 635 640 Ser Asp Ile Lys ThrLeu Ala Lys Val Ser Ala Ala Asn Thr Asp Arg 645 650 655 Ile Ala Lys AsnLys Ala Glu Ala Asp Ala Ser Phe Glu Thr Leu Thr 660 665 670 Lys Asn GlnAsn Thr Leu Ile Glu Gln Gly Glu Ala Leu Val Glu Gln 675 680 685 Asn LysAla Ile Asn Gln Glu Leu Glu Gly Phe Ala Ala His Ala Asp 690 695 700 ValGln Asp Lys Gln Ile Leu Gln Asn Gln Ala Asp Ile Thr Ala Asn 705 710 715720 Lys Thr Ala Ile Glu Gln Asn Ile Asn Arg Thr Val Ala Asn Gly Phe 725730 735 Glu Ile Glu Lys Asn Lys Ala Gly Ile Ala Thr Asn Lys Gln Glu Leu740 745 750 Ile Leu Gln His Asp Arg Leu Asn Arg Ile Asn Glu Thr Asn AsnArg 755 760 765 Gln Asp Gln Lys Ile Asp Gln Leu Gly Tyr Ala Leu Lys GluGln Gly 770 775 780 Gln His Phe Asn Asn Arg Ile Ser Ala Val Glu Arg GlnThr Ala Gly 785 790 795 800 Gly Ile Ala Asn Ala Ile Ala Ile Ala Thr LeuPro Ser Pro Ser Arg 805 810 815 Ala Gly Glu His His Val Leu Phe Gly SerGly Tyr His Asn Gly Gln 820 825 830 Ala Ala Val Ser Leu Gly Ala Ala GlyLeu Ser Asp Thr Gly Lys Ser 835 840 845 Thr Tyr Lys Ile Gly Leu Ser TrpSer Asp Ala Gly Gly Leu Ser Gly 850 855 860 Gly Val Gly Gly Ser Tyr ArgTrp Lys 865 870 14 3292 DNA Moraxella catarrhalis 14 gtaaatgactgatgagtgtc tatttaatga aagatacaat atataaaagt tgactatagc 60 gatgcaatacagtaaaattt gttacggcta aacataacga cggtccaaga tggcggatat 120 cgccatttaccaacctgata atcagtttga tagccattag cgatggcatc aagttgtgtt 180 gttgtattgtcatataaacg gtaaatttgg tttggtggat gccccatctg atttaccgtc 240 cccctaataagtgagagggg gggggagacc ccagtcattt attaggagac taagatgaac 300 aaaatttataaagtgaaaaa aaatgccgca ggtcacttgg tggcatgttc tgaatttgcc 360 aaaggccataccaagaaggc agttttgggc agtttattga ttgttggaat attgggtatg 420 gcaacgacagcatctgcaca acaaacaatc gcacgccaag gcaaaggcat gcactctatc 480 atcggtggtggcaatgacaa cgaagccaac ggcgattact ctaccgtcag tggtggcgat 540 tataacgaagccaaaggcga tagctctacc atcggtggtg gctattataa cgaagccaac 600 ggcgatagctctaccatcgg tggtggcttt tataacgaag ccaaaggcga gagctctacc 660 atcggtggtggcgataacaa ctcagccaca ggcatgtact ctaccatcgg tggtggcgat 720 aacaactcagccacaggcag gtactctacc atcgcagggg gttggcttaa ccaagctaca 780 ggtcatagctcaacggttgc agggggttgg cttaaccaag ctacaaacga gaattctacc 840 gttggtggcggcaggtttaa ccaagctaca ggtcgtaact caacggttgc agggggctat 900 aaaaacaaagccacaggcgt agactctacc atcgcagggg gcaggaataa ccaagccaac 960 ggtataggttcatttgcagc aggtatagac aaccaagcca atgccaacaa caccgtcgct 1020 ctaggtaacaagaacatcat caaaggtaaa gactcagtag ccatcggctc taataatacc 1080 gttgaaactggcaaagaaaa tgtctttatt cttggctcta acacaaaaga tgcacatagt 1140 aactcagtgctactgggtaa tgagaccact ggcaaagcag cgaccactgt tgagaatgcc 1200 aaagtgggtggtctaagcct aacaggattt gtaggtgcat caaaagctaa tactaataat 1260 ggtactgtatctgtcggtaa gcagggtaaa gagcgtcaaa tcgttaatgt tggtgcaggt 1320 cagatccgtgctgattcaac agatgctgtt aatggctcac agctacatgc tttggccaca 1380 gctgtcgatgcagaatttag aacactcacc caaactcaaa atgctttgat tgagcaaggt 1440 gaagccatcaatcaagagct tgaaggtttg gcagattata caaatgctca agatgagaaa 1500 attctaaaaaaccaaactga catcactgcc aataaaactg ctattgagca aaattttaat 1560 agaactgttaccaatgggtt tgagattgag aaaaataaag ctggtattgc taaaaaccaa 1620 gcggatatccaaacacttga aaacgatgtc ggaaaagaac tattaaatct aagcggtcgc 1680 ctgcttgatcaaaaagcaga tattgataat aacatcaaca atatctatga gctggcacaa 1740 cagcaagatcagcatagctc tgatatcaaa acacttaaaa acaatgtcga agaaggtttg 1800 ttggatctaagcggtcgcct cattgatcaa aaagcagatc ttacgaaaga catcaaagca 1860 cttgaaaacaatgtcgaaga aggtttattg gatctaagcg gtcgcctcat tgatcaaaaa 1920 gcagatattgctaaaaacca agcagacatc caagatttgg ccgcttacaa cgagctacaa 1980 gaccagtatgctcaaaagca aaccgaagcg attgacgctc taaataaagc aagctctgcc 2040 aatactgatcgtattgctac tgctgaattg ggtatcgctg agaacaaaaa agacgctcag 2100 atcgccaaagcacaagccaa tgaaaataaa gacggcattg ctaaaaacca agcagatatt 2160 gctaataacatcaaaaatat ctatgagctg gcacaacagc aagatcagca tagctctgat 2220 atcaaaaccttggcaaaagt aagtgctgcc aatactgatc gtattgctaa aaacaaagct 2280 gaagctgatgcaagttttga aacgctcacc aaaaatcaaa atactttgat tgagcaaggt 2340 gaagcattggttgagcaaaa taaagccatc aatcaagagc ttgaagggtt tgcggctcat 2400 gcagatgttcaagataagca aattttacaa aaccaagctg atatcactgc caataagacc 2460 gctattgaacaaaatatcaa tagaactgtt gccaatgggt ttgagattga gaaaaataaa 2520 gctggtattgctaccaataa gcaagagctt attcttcaac atgatcgatt aaatcgaatt 2580 aatgagacaaataatcgtca ggatcagaag attgatcaat taggttatgc actaaaagag 2640 cagggtcagcattttaataa tcgtattagt gctgttgagc gtcaaacagc tggaggtatt 2700 gcaaatgctatcgcaattgc aactttacca tcgcccagta gagcaggtga gcatcatgtc 2760 ttatttggttcaggttatca caatggtcaa gctgcggtat cattgggtgc ggctgggtta 2820 agtgatacaggaaaatcaac ttataagatt ggtctaagct ggtcagatgc aggtggatta 2880 tctggtggtgttggtggtag ttaccgctgg aaatagagcc taaatttaac tgctgtatca 2940 aaaaatatggtctgtataaa cagaccatat ttttatctaa aaacttatct taacttttat 3000 gaagcatcataagccaaagc tgagtaataa taagagatgt taaaataaga gatgttaaaa 3060 ctgctaaacaatcggcttac gacgataaaa taaaatacct ggaatggaca gccccaaaac 3120 caatgctgagatgataaaaa tcgcctcaaa aaaatgacgc atcataacga taaataaatc 3180 catatcaaatccaaaatagc caatttgtac catgctaacc atggctttat aggcagcgat 3240 tcccggcatcatacaaatca agctaggtac aatcaaggct ttaggcggca gg 3292 15 889 PRT Moraxellacatarrhalis 15 Val Asn Lys Ile Tyr Lys Val Lys Lys Asn Ala Ala Gly HisLeu Val 1 5 10 15 Ala Cys Ser Glu Phe Ala Lys Gly His Thr Lys Lys AlaVal Leu Gly 20 25 30 Ser Leu Leu Ile Val Gly Ala Leu Gly Met Ala Thr ThrAla Ser Ala 35 40 45 Gln Pro Leu Val Ser Thr Asn Lys Pro Asn Gln Gln ValLys Gly Tyr 50 55 60 Trp Ser Ile Ile Gly Ala Gly Arg His Asn Asn Val GlyGly Ser Ala 65 70 75 80 His His Ser Gly Ile Leu Gly Gly Trp Lys Asn ThrVal Asn Gly Tyr 85 90 95 Thr Ser Ala Ile Val Gly Gly Tyr Gly Asn Glu ThrGln Gly Asp Tyr 100 105 110 Thr Phe Val Gly Gly Gly Tyr Lys Asn Leu AlaLys Gly Asn Tyr Thr 115 120 125 Phe Val Gly Gly Gly Tyr Lys Asn Leu AlaGlu Gly Asp Asn Ala Thr 130 135 140 Ile Ala Gly Gly Phe Ala Asn Leu AlaGlu Gly Asp Asn Ala Thr Ile 145 150 155 160 Ala Gly Gly Phe Glu Asn ArgAla Glu Gly Ile Asp Ser Val Val Ser 165 170 175 Gly Gly Tyr Ala Asn GlnAla Thr Gly Glu Ser Ser Thr Val Ala Gly 180 185 190 Gly Ser Asn Asn LeuAla Glu Gly Lys Ser Ser Ala Ile Gly Gly Gly 195 200 205 Arg Gln Asn GluAla Ser Gly Asp Arg Ser Thr Val Ser Gly Gly Tyr 210 215 220 Asn Asn LeuAla Glu Gly Lys Ser Ser Ala Ile Gly Gly Gly Glu Phe 225 230 235 240 AsnLeu Ala Leu Gly Asn Asn Ala Thr Ile Ser Gly Gly Arg Gln Asn 245 250 255Glu Ala Ser Gly Asp Arg Ser Thr Val Ala Gly Gly Glu Gln Asn Gln 260 265270 Ala Ile Gly Lys Tyr Ser Thr Ile Ser Gly Gly Arg Gln Asn Glu Ala 275280 285 Ser Gly Asp Arg Ser Thr Val Ala Gly Gly Glu Gln Asn Gln Ala Ile290 295 300 Gly Lys Tyr Ser Thr Val Ser Gly Gly Tyr Arg Asn Gln Ala ThrGly 305 310 315 320 Lys Gly Ser Phe Ala Ala Gly Ile Asp Asn Lys Ala AsnAla Asp Asn 325 330 335 Ala Val Ala Leu Gly Asn Lys Asn Thr Ile Glu GlyGlu Asn Ser Val 340 345 350 Ala Ile Gly Ser Asn Asn Thr Val Lys Lys AsnGln Lys Asn Val Phe 355 360 365 Ile Leu Gly Ser Asn Thr Asp Thr Lys AspAla Gln Ser Gly Ser Val 370 375 380 Leu Leu Gly Asp Asn Thr Ser Gly LysAla Ala Thr Ala Val Glu Asp 385 390 395 400 Ala Thr Val Gly Asp Leu SerLeu Thr Gly Phe Ala Gly Val Ser Lys 405 410 415 Ala Asn Ser Gly Thr ValSer Val Gly Ser Glu Gly Lys Glu Arg Gln 420 425 430 Ile Val His Val GlyAla Gly Arg Ile Ser Asn Asp Ser Thr Asp Ala 435 440 445 Val Asn Gly SerGln Leu Tyr Ala Leu Ala Ala Ala Val Asp Asp Asn 450 455 460 Gln Tyr AspIle Glu Lys Asn Gln Asp Asp Ile Ala Lys Asn Gln Ala 465 470 475 480 AspIle Ala Lys Asn Gln Ala Asp Ile Gln Thr Leu Glu Asn Asp Val 485 490 495Gly Lys Glu Leu Leu Asn Leu Ser Gly Arg Leu Ile Asp Gln Lys Ala 500 505510 Asp Ile Asp Asn Asn Ile Asn His Ile Tyr Glu Leu Ala Gln Gln Gln 515520 525 Asp Gln His Ser Ser Asp Ile Lys Thr Leu Lys Lys Asn Val Glu Glu530 535 540 Gly Leu Leu Glu Leu Ser Gly His Leu Ile Asp Gln Lys Ala AspLeu 545 550 555 560 Thr Lys Asp Ile Lys Ala Leu Glu Ser Asn Val Glu GluGly Leu Leu 565 570 575 Asp Leu Ser Gly Arg Leu Ile Asp Gln Lys Ala AspIle Ala Gln Asn 580 585 590 Gln Ala Asn Ile Gln Asp Leu Ala Ala Tyr AsnGlu Leu Gln Asp Gln 595 600 605 Tyr Ala Gln Lys Gln Thr Glu Ala Ile AspAla Leu Asn Lys Ala Ser 610 615 620 Ser Glu Asn Thr Gln Asn Ile Glu AspLeu Ala Ala Tyr Asn Glu Leu 625 630 635 640 Gln Asp Ala Tyr Ala Lys GlnGln Thr Glu Ala Ile Asp Ala Leu Asn 645 650 655 Lys Ala Ser Ser Glu AsnThr Gln Asn Ile Ala Lys Asn Gln Ala Asp 660 665 670 Ile Ala Asn Asn IleAsn Asn Ile Tyr Glu Leu Ala Gln Gln Gln Asp 675 680 685 Gln His Ser SerAsp Ile Lys Thr Leu Ala Lys Ala Ser Ala Ala Asn 690 695 700 Thr Asp ArgIle Ala Lys Asn Lys Ala Asp Ala Asp Ala Ser Phe Glu 705 710 715 720 ThrLeu Thr Lys Asn Gln Asn Thr Leu Ile Glu Lys Asp Lys Glu His 725 730 735Asp Lys Leu Ile Thr Ala Asn Lys Thr Ala Ile Asp Ala Asn Lys Ala 740 745750 Ser Ala Asp Thr Lys Phe Ala Ala Thr Ala Asp Ala Ile Thr Lys Asn 755760 765 Gly Asn Ala Ile Thr Lys Asn Ala Lys Ser Ile Thr Asp Leu Gly Thr770 775 780 Lys Val Asp Gly Phe Asp Gly Arg Val Thr Ala Leu Asp Thr LysVal 785 790 795 800 Asn Ala Phe Asp Gly Arg Ile Thr Ala Leu Asp Ser LysVal Glu Asn 805 810 815 Gly Met Ala Ala Gln Ala Ala Leu Ser Gly Leu PheGln Pro Tyr Ser 820 825 830 Val Gly Lys Phe Asn Ala Thr Ala Ala Leu GlyGly Tyr Gly Ser Lys 835 840 845 Ser Ala Val Ala Ile Gly Ala Gly Tyr ArgVal Asn Pro Asn Leu Ala 850 855 860 Phe Lys Ala Gly Ala Ala Ile Asn ThrSer Gly Asn Lys Lys Gly Ser 865 870 875 880 Tyr Asn Ile Gly Val Asn TyrGlu Phe 885 16 4228 DNA Moraxella catarrhalis 16 gccgcaccct gaccgagacgctccgccaaa tcgatgcgtc ggtgtactat gccccgaccg 60 agctatgcac ggataatggtgcgatgatcg cctatgctgg cttttgtcgg ctaagccgtg 120 gacagtcgga tgacttggtggttcgctgta ttccccgatg ggatatgacg acgcttggta 180 tcgaatatga taattaggctgtggtatttg agttttgagt aatgtaccta ctaccactaa 240 tttatcatac aatacataaacataaaaaac atcggtattg ttaaaaaaca atacccaagt 300 taaaatagct caatactttaccatagcaca aagaaacttg tgaacgaaac atttaataat 360 tgcccaaaat gttactgcacacactttgta aaagcaggct tgggcaatgg caaacaacga 420 tacaaatgca aaggttgccatcactatttt tctgtgaagc aacgaagcaa ccaaaaaagt 480 aatgacatta aaaaaacaagccattgatac aaacagtaaa caaatcttag gctttgtctg 540 tggtaaaaca gacactaacacctttaaacg actttatcag cagttaaata cccatagcat 600 tcaactgttt tttagtgactactggaaatc ttatcgtcaa gtcattttaa agccaaaaca 660 tataacaagc aaagctcaaacttttaccat agagggctat aatagtctca ttaggcattt 720 catagcaaga tttacaagaaagtcaaagtg ttattctaaa tccgaaaaaa tgatagaaaa 780 cacgttgaat ttattatttgctaagtggaa tggtagctta agatatgtat tttaatttaa 840 caatgccaaa aacatcaattacagtaagat tttaggcgtt ttgcagttgc tactttagta 900 aagctttgtt atactagctgttagtatact caagcttgtt tgtgtttgag ctatatttat 960 tttatagcag tagttggttataaaatataa ataaagctaa gctcgagggt ttggtaatgg 1020 ttttttatgt ttataataccaacagagtct atacagctaa aatagctaat accttaggtg 1080 tattacaagt aaaaatcctttggttaatca gggggtgtat tatatgtata tttcctttgt 1140 atttggttat agcaatcccttggtaagaaa tcatatctat tttttattgt tcaattattt 1200 aggagactaa ggtgaacaaaatttataaag tgaaaaaaaa tgccgcaggt cacttggtgg 1260 catgttctga atttgccaaaggccatacca aaaaggcagt tttgggcagt ttattgattg 1320 ttggggcgtt gggcatggcaacgacggcgt ctgcacagcc attagtaagt acaaataagc 1380 ctaatcagca ggtaaagggttattggtcta ttattggtgc aggtcgtcat aataacgtag 1440 gtggatccgc tcatcactcagggattcttg gtggttggaa aaatacagtc aatggctata 1500 cctcagccat tgtaggtggttatggtaacg aaactcaggg tgattataca ttcgtcggtg 1560 gtggttataa aaacttggcaaagggtaatt atacattcgt cggtggtggt tataaaaact 1620 tggcagaggg tgataatgcaaccatcgctg gtggttttgc aaacttggca gagggtgata 1680 atgcaaccat cgctggtggttttgaaaacc gtgcagaggg tatcgactca gtagtttctg 1740 gtggttatgc caaccaagctacaggagaaa gctcaaccgt cgcaggtggt tctaataacc 1800 tagcagaggg caaaagctcagccattggtg gtggccgtca aaatgaggcg tctggtgacc 1860 gatctactgt ctcaggtggttataataacc tagcagaggg caaaagctca gccattggtg 1920 gcggtgagtt taacttagcattagggaata acgctaccat tagtggtggc cgtcaaaatg 1980 aggcgtctgg tgaccgatctactgtcgcag gtggtgaaca aaaccaagcc ataggcaagt 2040 attctaccat tagtggtggccgtcaaaatg aggcgtctgg tgaccgatct actgtcgcag 2100 gtggtgaaca aaaccaagccataggcaagt attctaccgt tagtggtggc tatcgaaacc 2160 aagccacagg taaaggttcatttgcagcag gtatagataa caaagccaat gccgacaacg 2220 ccgtcgctct aggtaacaagaacaccatcg aaggtgaaaa ctcagtagcc atcggctcta 2280 ataataccgt taaaaaaaatcaaaaaaatg tctttattct tggctctaac acagacacaa 2340 aagatgcaca aagcggctcagtactgctag gtgataatac ctctggtaaa gcagcgaccg 2400 ctgttgagga tgccacagtgggtgatctaa gcctaacagg atttgcaggc gtatcaaaag 2460 ctaatagtgg tactgtatctgtcggtagtg agggtaaaga gcgtcaaatc gttcatgttg 2520 gtgcaggtcg gatcagtaatgattcaacag atgctgttaa tggctcacag ctatatgctt 2580 tggccgcagc tgttgatgacaaccaatatg acattgaaaa aaaccaagat gacattgcta 2640 aaaaccaagc tgacattgctaaaaaccaag ctgacatcca aacacttgaa aacgatgtcg 2700 gaaaagaact attaaatctaagcggtcgcc tcattgatca aaaagcagat attgataata 2760 acatcaacca tatctatgagctggcacaac agcaagatca gcatagctct gatatcaaaa 2820 cacttaaaaa aaatgtcgaagaaggtttgt tggagctaag cggtcacctc attgatcaaa 2880 aagcagatct tacaaaagacatcaaagcac ttgaaagcaa tgtcgaagaa ggtttgttgg 2940 atctaagcgg tcgcctcattgatcaaaaag cagatattgc tcaaaaccaa gctaacatcc 3000 aagatttggc tgcttacaacgagctacaag accagtatgc tcaaaagcaa accgaagcga 3060 ttgacgctct aaataaagcaagctctgaga atacacaaaa catcgaagat ctggccgctt 3120 acaacgagct acaagatgcctatgccaaac agcaaaccga agccattgac gctctaaata 3180 aagcaagctc tgagaatacacaaaacattg ctaaaaacca agcggatatt gctaataaca 3240 tcaacaatat ctatgagctagcacaacagc aagatcagca tagctctgat atcaaaacct 3300 tggcaaaagc aagtgctgccaatactgatc gtattgctaa aaacaaagcc gatgctgatg 3360 caagttttga aacgctcaccaaaaatcaaa atactttgat tgaaaaagat aaagagcatg 3420 acaaattaat tactgcaaacaaaactgcga ttgatgccaa taaagcatct gcggatacca 3480 agtttgcagc gacagcagacgccattacca aaaatggaaa tgctatcact aaaaacgcaa 3540 aatctatcac tgatttgggtactaaagtgg atggttttga cggtcgtgta actgcattag 3600 acaccaaagt caatgcctttgatggtcgta tcacagcttt agacagtaaa gttgaaaacg 3660 gtatggctgc ccaagctgccctaagtggtc tattccagcc ttatagcgtt ggtaagttta 3720 atgcgaccgc tgcacttggtggctatggct caaaatctgc ggttgctatc ggtgctggct 3780 atcgtgtgaa tccaaatctggcgtttaaag ctggtgcggc gattaatacc agtggcaata 3840 aaaaaggctc ttataacatcggtgtgaatt acgagttcta attgtctatc atcaccaaaa 3900 aaagcagtca gtttactggctgctttttta tgggtttttg tggcttttgg ttgtgagtga 3960 tggataaaag cttgtcaagcgattgatgaa tatcaataaa tgattggtaa atatcaataa 4020 agcggtttag ggtttttggatatcttttaa taagtttaaa aacccctgca taaaataaag 4080 ctggcatcag agctgcgaagtagcggcata cagctggcaa tgcacgcctg tgcctagggg 4140 gcgtgagacc acccagcctttgcgttcgta ttctaaaatt acccaatcag gcagagcggc 4200 aactccatgt tcggaggcgaccagctga 4228 17 7 PRT Moraxella catarrhalis 17 Ala Gln Gln Gln Asp GlnHis 1 5 18 10 PRT Moraxella catarrhalis 18 Tyr Glu Leu Ala Gln Gln GlnAsp Gln His 1 5 10 19 10 PRT Moraxella catarrhalis 19 Tyr Asp Leu AlaGln Gln Gln Asp Gln His 1 5 10 20 22 DNA Moraxella catarrhalis 20gacgctcaac agcactaata cg 22 21 19 DNA Moraxella catarrhalis 21ccaagctgat atcactacc 19 22 18 DNA Moraxella catarrhalis 22 tcaatgcctttgatggtc 18 23 21 DNA Moraxella catarrhalis 23 tgtatgccgc tactcgcagc t21 24 14 PRT Moraxella catarrhalis MOD_RES (2)..(13) Xaa = any 24 AsnXaa Ala Xaa Xaa Tyr Ser Xaa Ile Gly Gly Gly Xaa Asn 1 5 10 25 4 PRTMoraxella catarrhalis 25 Gln Ala Asp Ile 1 26 30 PRT Moraxellacatarrhalis 26 Ala Ala Gln Ala Ala Leu Ser Gly Leu Phe Val Pro Tyr SerVal Gly 1 5 10 15 Lys Phe Asn Ala Thr Ala Ala Leu Gly Gly Tyr Gly SerLys 20 25 30 27 13 PRT Moraxella catarrhalis 27 Gly Lys Ile Thr Lys AsnAla Ala Arg Gln Glu Asn Gly 1 5 10 28 9 PRT Moraxella catarrhalis 28 ValIle Gly Asp Leu Gly Arg Lys Val 1 5 29 10 PRT Moraxella catarrhalisMOD_RES (4) Xaa = any 29 Ala Leu Glu Xaa Asn Val Glu Glu Gly Leu 1 5 1030 14 PRT Moraxella catarrhalis MOD_RES (11)..(12) Xaa = any 30 Ala LeuGlu Ser Asn Val Glu Glu Gly Leu Xaa Xaa Leu Ser 1 5 10 31 7 PRTMoraxella catarrhalis 31 Ala Leu Glu Phe Asn Gly Glu 1 5 32 9 PRTMoraxella catarrhalis MOD_RES (7) Xaa = any 32 Ser Ile Thr Asp Leu GlyXaa Lys Val 1 5 33 15 PRT Moraxella catarrhalis MOD_RES (13)..(15) Xaa =any 33 Ser Ile Thr Asp Leu Gly Thr Ile Val Asp Gly Phe Xaa Xaa Xaa 1 510 15 34 10 PRT Moraxella catarrhalis 34 Ser Ile Thr Asp Leu Gly Thr IleVal Asp 1 5 10 35 20 PRT Moraxella catarrhalis MOD_RES (5)..(12) Xaa =any 35 Val Asp Ala Leu Xaa Thr Lys Val Asn Ala Leu Asp Xaa Lys Val Asn 15 10 15 Ser Asp Xaa Thr 20 36 15 PRT Moraxella catarrhalis 36 Leu LeuAla Glu Gln Gln Leu Asn Gly Lys Thr Leu Thr Pro Val 1 5 10 15 37 14 PRTMoraxella catarrhalis 37 Ala Lys His Asp Ala Ala Ser Thr Glu Lys Gly LysMet Asp 1 5 10 38 15 PRT Moraxella catarrhalis 38 Ala Leu Glu Ser AsnVal Glu Glu Gly Leu Leu Asp Leu Ser Gly 1 5 10 15 39 12 PRT Moraxellacatarrhalis 39 Asn Gln Asn Thr Leu Ile Glu Lys Thr Ala Asn Lys 1 5 10 409 PRT Moraxella catarrhalis 40 Ile Asp Lys Asn Glu Tyr Ser Ile Lys 1 541 8 PRT Moraxella catarrhalis 41 Ser Ile Thr Asp Leu Gly Thr Lys 1 5 428 PRT Moraxella catarrhalis 42 Asn Gln Asn Thr Leu Ile Glu Lys 1 5 43 12PRT Moraxella catarrhalis 43 Ala Leu His Glu Gln Gln Leu Glu Thr Leu ThrLys 1 5 10 44 4 PRT Moraxella catarrhalis 44 Asn Ser Ser Asp 1 45 14 PRTMoraxella catarrhalis 45 Asn Lys Ala Asp Ala Asp Ala Ser Phe Glu Thr LeuThr Lys 1 5 10 46 10 PRT Moraxella catarrhalis 46 Phe Ala Ala Thr AlaIle Ala Lys Asp Lys 1 5 10 47 12 PRT Moraxella catarrhalis 47 Lys AlaSer Ser Glu Asn Thr Gln Asn Ile Ala Lys 1 5 10 48 6 PRT Moraxellacatarrhalis 48 Arg Leu Leu Asp Gln Lys 1 5 49 12 PRT Moraxellacatarrhalis MOD_RES (12) Xaa = any 49 Ala Ala Thr Ala Asp Ala Ile ThrLys Asn Gly Xaa 1 5 10 50 10 PRT Moraxella catarrhalis MOD_RES (4)..(8)Xaa = any 50 Ala Lys Ala Xaa Ala Ala Asn Xaa Asp Arg 1 5 10 51 22 PRTMoraxella catarrhalis 51 Asn Gln Ala Asp Ile Ala Gln Asn Gln Thr Asp IleGln Asp Leu Ala 1 5 10 15 Ala Tyr Asn Glu Leu Gln 20 52 21 PRT Moraxellacatarrhalis 52 Asn Gln Ala Asp Ile Ala Asn Asn Ile Asn Asn Ile Tyr GluLeu Ala 1 5 10 15 Gln Gln Gln Asp Gln 20 53 13 PRT Moraxella catarrhalis53 Tyr Asn Glu Arg Gln Thr Glu Ala Ile Asp Ala Leu Asn 1 5 10 54 13 PRTMoraxella catarrhalis 54 Ile Leu Gly Asp Thr Ala Ile Val Ser Asn Ser GlnAsp 1 5 10 55 17 PRT Moraxella catarrhalis 55 Lys Ala Leu Glu Ser AsnVal Glu Glu Gly Leu Leu Asp Leu Ser Gly 1 5 10 15 Arg 56 21 PRTMoraxella catarrhalis 56 Ala Leu Glu Ser Asn Val Glu Glu Gly Leu Leu GluLeu Ser Gly Arg 1 5 10 15 Thr Ile Asp Gln Arg 20 57 22 PRT Moraxellacatarrhalis MOD_RES (11) Xaa = any 57 Asn Gln Ala His Ile Ala Asn AsnIle Asn Xaa Ile Tyr Glu Leu Ala 1 5 10 15 Gln Gln Gln Asp Gln Lys 20 5822 PRT Moraxella catarrhalis 58 Asn Gln Ala Asp Ile Ala Gln Asn Gln ThrAsp Ile Gln Asp Leu Ala 1 5 10 15 Ala Tyr Asn Glu Leu Gln 20 59 12 PRTMoraxella catarrhalis 59 Ala Thr His Asp Tyr Asn Glu Arg Gln Thr Glu Ala1 5 10 60 12 PRT Moraxella catarrhalis 60 Lys Ala Ser Ser Glu Asn ThrGln Asn Ile Ala Lys 1 5 10 61 23 PRT Moraxella catarrhalis 61 Met IleLeu Gly Asp Thr Ala Ile Val Ser Asn Ser Gln Asp Asn Lys 1 5 10 15 ThrGln Leu Lys Phe Tyr Lys 20 62 14 PRT Moraxella catarrhalis MOD_RES(12)..(13) Xaa = any 62 Ala Gly Asp Thr Ile Ile Pro Leu Asp Asp Asp XaaXaa Pro 1 5 10 63 10 PRT Moraxella catarrhalis MOD_RES (8) Xaa = any 63Leu Leu His Glu Gln Gln Leu Xaa Gly Lys 1 5 10 64 6 PRT Moraxellacatarrhalis MOD_RES (5) Xaa = any 64 Ile Phe Phe Asn Xaa Gly 1 5 65 23PRT Moraxella catarrhalis 65 Asn Asn Ile Asn Asn Ile Tyr Glu Leu Ala GlnGln Gln Asp Gln His 1 5 10 15 Ser Ser Asp Ile Lys Thr Leu 20 66 21 DNAMoraxella catarrhalis 66 ggtgcaggtc agatcagtga c 21 67 18 DNA Moraxellacatarrhalis 67 gccaccaacc aagctgac 18 68 21 DNA Moraxella catarrhalis 68agcggtcgcc tgcttgatca g 21 69 21 DNA Moraxella catarrhalis 69 ctgatcaagcaggcgaccgc t 21 70 20 DNA Moraxella catarrhalis 70 caagatctgg ccgcttacaa20 71 20 DNA Moraxella catarrhalis 71 ttgtaagcgg ccagatcttg 20 72 18 DNAMoraxella catarrhalis 72 tgcatgagcc gcaaaccc 18 73 9 PRT Moraxellacatarrhalis 73 Leu Leu Ala Glu Gln Gln Leu Asn Gly 1 5 74 10 PRTMoraxella catarrhalis 74 Ala Leu Glu Ser Asn Val Glu Glu Gly Leu 1 5 1075 14 PRT Moraxella catarrhalis 75 Ala Leu Glu Ser Asn Val Glu Glu GlyLeu Leu Asp Leu Ser 1 5 10 76 3788 PRT Moraxella catarrhalis MOD_RES(1036)..(3786) Xaa = any 76 Thr His Glu Phe Ile Arg Ser Thr Ser Glu GlnGlu Asn Cys Glu Ser 1 5 10 15 Ala Arg Glu Asn Thr His Glu Asp Ile SerLys Glu Thr Thr Glu Ser 20 25 30 Ala Asn Asp Ala Thr Leu Glu Ala Ser ThrSer Met Glu Phe Thr His 35 40 45 Glu Ser Glu Ala Leu Arg Glu Ala Asp TyrAsn Thr His Glu Thr Asp 50 55 60 Arg Ile Val Glu Cys His Glu Cys Lys TrpIle Thr His Gly Glu Arg 65 70 75 80 Arg Ile Thr His Glu Ser Glu Ser GluGln Glu Asn Cys Glu Ser Ala 85 90 95 Arg Glu Asn Ala Met Glu Asp Asn ThrHis Glu Asp Ile Ser Lys Glu 100 105 110 Thr Thr Glu Ser Ala Asn Asp HisAla Arg Asp Cys Pro Ile Glu Ser 115 120 125 Ala Thr Thr Ala Cys His GluAsp Ala Ser Phe Leu Leu Trp Ser Ala 130 135 140 Asn Asp Asp Asn Thr HisAla Val Glu Ala Asn Tyr Ser Pro Glu Cys 145 150 155 160 Ile Ala Leu CysHis Ala Arg Ala Cys Thr Glu Arg Ser Asp Asn Thr 165 170 175 Thr Arg AlaAsn Ser Leu Ala Thr Glu Ala Asn Tyr Ser Glu Gln Glu 180 185 190 Asn CysGlu Ser Thr His Ala Thr Ile Ser Thr His Glu Arg Glu Ile 195 200 205 SerAsn Ser Thr Ala Arg Thr Cys Asp Asn Ser Glu Gln Ile Asp Asn 210 215 220Phe Ile Leu Glu Asn Ala Met Glu Thr Tyr Pro Glu Ser Thr Arg Ala 225 230235 240 Asn Asp Thr Pro Leu Gly Tyr Ser Glu Gln Ile Asp Asn Glu Ser Pro245 250 255 Ala Ala Ala Pro Arg Thr Glu Ile Asn Asn Ala Leu Ile Asn GluAla 260 265 270 Arg Ser Glu Gln Ile Asp Asn Glu Ser Pro Ala Asn Ala AspAsn Ala 275 280 285 Asp Asx Leu Glu Leu Ile Asn Glu Ala Arg Ser Glu GlnIle Asp Asn 290 295 300 Glu Ser Pro Ala Ala Ala Pro Arg Thr Glu Ile AsnAsn Ala Leu Ile 305 310 315 320 Asn Glu Ala Arg Ser Glu Gln Ile Asp AsnGlu Ser Pro Ala Asn Ala 325 330 335 Asp Asn Ala Asp Asx Leu Glu Leu IleAsn Glu Ala Arg Ser Glu Gln 340 345 350 Ile Asp Asn Glu Ser Pro Ala AlaAla Pro Ala Thr Pro Arg Thr Glu 355 360 365 Ile Asn Asn Ala Leu Ile AsnGlu Ala Arg Ser Glu Gln Ile Asp Asn 370 375 380 Glu Ser Pro Ala Asn AlaPro Ala Thr Asp Asn Ala Asp Asx Leu Glu 385 390 395 400 Leu Ile Asn GluAla Arg Ser Glu Gln Ile Asp Asn Glu Ser Pro Ala 405 410 415 Ala Ala ProAla Thr Pro Arg Thr Glu Ile Asn Asn Ala Leu Ile Asn 420 425 430 Glu AlaArg Ser Glu Gln Ile Asp Asn Glu Ser Pro Ala Asn Ala Pro 435 440 445 AlaThr Asp Asn Ala Asp Asx Leu Glu Leu Ile Asn Glu Ala Arg Ser 450 455 460Glu Gln Ile Asp Asn Thr Thr Ala Ser Pro Ala Ala Ala Pro Ala Thr 465 470475 480 Pro Arg Thr Glu Ile Asn Asn Ala Leu Ile Asn Glu Ala Arg Ser Glu485 490 495 Gln Ile Asp Asn Thr Thr Ala Ser Pro Ala Asn Ala Pro Ala ThrAsp 500 505 510 Asn Ala Asp Asx Leu Glu Leu Ile Asn Glu Ala Arg Ser GluGln Ile 515 520 525 Asp Asn Thr Thr Ala Ser Pro Ala Ala Ala Pro Ala ThrPro Arg Thr 530 535 540 Glu Ile Asn Asn Ala Leu Ile Asn Glu Ala Arg SerGlu Gln Ile Asp 545 550 555 560 Asn Thr Thr Ala Ser Pro Ala Asn Ala ProAla Thr Asp Asn Ala Asp 565 570 575 Asx Leu Glu Leu Ile Asn Glu Ala ArgSer Glu Gln Ile Asp Asn Thr 580 585 590 Thr Ala Ser Pro Ala Ala Ala ProAla Thr Pro Arg Thr Glu Ile Asn 595 600 605 Asn Ala Leu Ile Asn Glu AlaArg Ser Glu Gln Ile Asp Asn Thr Thr 610 615 620 Ala Ser Pro Ala Asn AlaPro Ala Thr Asp Asn Ala Asp Asx Leu Glu 625 630 635 640 Leu Ile Asn GluAla Arg Ser Glu Gln Ile Asp Asn Thr Thr Ala Ser 645 650 655 Pro Ala AlaAla Pro Ala Thr Pro Arg Thr Glu Ile Asn Asn Ala Leu 660 665 670 Ile AsnGlu Ala Arg Ser Glu Gln Ile Asp Asn Thr Thr Ala Ser Pro 675 680 685 AlaAsn Ala Pro Ala Thr Asp Asn Ala Asp Asx Leu Glu Leu Ile Asn 690 695 700Glu Ala Arg Ser Glu Gln Ile Asp Asn Thr His Arg Gly His Ser Glu 705 710715 720 Gln Ile Asp Asn Ile Ser Gly Ile Val Glu Asn Asx Glu Leu Trp Ala725 730 735 Asn Asp Ala Arg Glu Asn Thr Asn Thr His Glu Asp Ile Ser LysGlu 740 745 750 Thr Thr Glu Ser Ser Glu Gln Glu Asn Cys Glu Ser Glu GlnIle Asp 755 760 765 Asn Thr Tyr Pro Glu Thr Pro Leu Gly Tyr Ser Thr ArgAla Asn Asp 770 775 780 Ser Pro Glu Cys Ile Ala Leu Ala Gln Gln Gln AspGln His Ser Glu 785 790 795 800 Gln Ile Asp Asn Pro Arg Thr Glu Ile AsnLeu Ile Asn Glu Ala Arg 805 810 815 Asn Ala Tyr Glu Leu Ala Gln Gln GlnAsp Gln His Ser Glu Gln Ile 820 825 830 Asp Asn Pro Arg Thr Glu Ile AsnLeu Ile Asn Glu Ala Arg Asn Ala 835 840 845 Tyr Asp Leu Ala Gln Gln GlnAsp Gln His Ser Glu Gln Ile Asp Asn 850 855 860 Pro Arg Thr Glu Ile AsnLeu Ile Asn Glu Ala Arg Asn Ala Gly Ala 865 870 875 880 Cys Gly Cys ThrCys Ala Ala Cys Ala Gly Cys Ala Cys Thr Ala Ala 885 890 895 Thr Ala CysGly Ser Glu Gln Ile Asp Asn Asp Asn Ala Leu Ile Asn 900 905 910 Glu AlaArg Asp Asx Leu Glu Cys Cys Ala Ala Gly Cys Thr Gly Ala 915 920 925 ThrAla Thr Cys Ala Cys Thr Ala Cys Cys Ser Glu Gln Ile Asp Asn 930 935 940Asp Asn Ala Leu Ile Asn Glu Ala Arg Asp Asx Leu Glu Thr Cys Ala 945 950955 960 Ala Thr Gly Cys Cys Thr Thr Thr Gly Ala Thr Gly Gly Thr Cys Ser965 970 975 Glu Gln Ile Asp Asn Asp Asn Ala Leu Ile Asn Glu Ala Arg AspAsx 980 985 990 Leu Glu Thr Gly Thr Ala Thr Gly Cys Cys Gly Cys Thr AlaCys Thr 995 1000 1005 Cys Gly Cys Ala Gly Cys Thr Ser Glu Gln Ile AspAsn Asp Asn Ala 1010 1015 1020 Leu Ile Asn Glu Ala Arg Asp Asx Leu GluAsn Xaa Ala Xaa Xaa Tyr 1025 1030 1035 1040 Ser Xaa Ile Gly Gly Gly XaaAsn Ser Glu Gln Ile Asp Asn Pro Arg 1045 1050 1055 Thr Glu Ile Asn LeuIle Asn Glu Ala Arg Asn Ala Xaa Ala Asn Tyr 1060 1065 1070 Ala Thr ProSer Ile Thr Ile Asn Ser Gln Ala Asp Ile Ser Glu Gln 1075 1080 1085 IleAsp Asn Pro Arg Thr Glu Ile Asn Leu Ile Asn Glu Ala Arg Asn 1090 10951100 Ala Ala Ala Gln Ala Ala Leu Ser Gly Leu Phe Val Pro Tyr Ser Val1105 1110 1115 1120 Gly Lys Phe Asn Ala Thr Ala Ala Leu Gly Gly Tyr GlySer Lys Ser 1125 1130 1135 Glu Gln Ile Asp Asn Pro Arg Thr Glu Ile AsnLeu Ile Asn Glu Ala 1140 1145 1150 Arg Asn Ala Gly Lys Ile Thr Lys AsnAla Ala Arg Gln Glu Asn Gly 1155 1160 1165 Ser Glu Gln Ile Asp Asn ProArg Thr Glu Ile Asn Leu Ile Asn Glu 1170 1175 1180 Ala Arg Asn Ala ValIle Gly Asp Leu Gly Arg Lys Val Ser Glu Gln 1185 1190 1195 1200 Ile AspAsn Pro Arg Thr Glu Ile Asn Leu Ile Asn Glu Ala Arg Asn 1205 1210 1215Ala Ala Leu Glu Xaa Asn Val Glu Glu Gly Leu Ser Glu Gln Ile Asp 12201225 1230 Asn Pro Arg Thr Glu Ile Asn Leu Ile Asn Glu Ala Arg Asn AlaXaa 1235 1240 1245 Ala Asn Tyr Ala Thr Pro Ser Ile Thr Ile Asn Ala LeuGlu Ser Asn 1250 1255 1260 Val Glu Glu Gly Leu Xaa Xaa Leu Ser Ser GluGln Ile Asp Asn Pro 1265 1270 1275 1280 Arg Thr Glu Ile Asn Leu Ile AsnGlu Ala Arg Asn Ala Xaa Ala Asn 1285 1290 1295 Tyr Ala Thr Pro Ser IleThr Ile Asn Ser Ala Leu Glu Phe Asn Gly 1300 1305 1310 Glu Ser Glu GlnIle Asp Asn Pro Arg Thr Glu Ile Asn Leu Ile Asn 1315 1320 1325 Glu AlaArg Asn Ala Ser Ile Thr Asp Leu Gly Xaa Lys Val Ser Glu 1330 1335 1340Gln Ile Asp Asn Pro Arg Thr Glu Ile Asn Leu Ile Asn Glu Ala Arg 13451350 1355 1360 Asn Ala Xaa Ala Asn Tyr Ala Thr Pro Ser Ile Thr Ile AsnSer Ile 1365 1370 1375 Thr Asp Leu Gly Thr Ile Val Asp Gly Phe Xaa XaaXaa Ser Glu Gln 1380 1385 1390 Ile Asp Asn Pro Arg Thr Glu Ile Asn LeuIle Asn Glu Ala Arg Asn 1395 1400 1405 Ala Xaa Ala Asn Tyr Ala Thr ProSer Ile Thr Ile Asn Ser Ser Ile 1410 1415 1420 Thr Asp Leu Gly Thr IleVal Asp Ser Glu Gln Ile Asp Asn Pro Arg 1425 1430 1435 1440 Thr Glu IleAsn Leu Ile Asn Glu Ala Arg Asn Ala Val Asp Ala Leu 1445 1450 1455 XaaThr Lys Val Asn Ala Leu Asp Xaa Lys Val Asn Ser Asp Xaa Thr 1460 14651470 Ser Glu Gln Ile Asp Asn Pro Arg Thr Glu Ile Asn Leu Ile Asn Glu1475 1480 1485 Ala Arg Asn Ala Xaa Ala Asn Tyr Ala Thr Pro Ser Ile ThrIle Asn 1490 1495 1500 Ser Leu Leu Ala Glu Gln Gln Leu Asn Gly Lys ThrLeu Thr Pro Val 1505 1510 1515 1520 Ser Glu Gln Ile Asp Asn Pro Arg ThrGlu Ile Asn Leu Ile Asn Glu 1525 1530 1535 Ala Arg Asn Ala Ala Lys HisAsp Ala Ala Ser Thr Glu Lys Gly Lys 1540 1545 1550 Met Asp Ser Glu GlnIle Asp Asn Pro Arg Thr Glu Ile Asn Leu Ile 1555 1560 1565 Asn Glu AlaArg Asn Ala Ala Leu Glu Ser Asn Val Glu Glu Gly Leu 1570 1575 1580 LeuAsp Leu Ser Gly Ser Glu Gln Ile Asp Asn Pro Arg Thr Glu Ile 1585 15901595 1600 Asn Leu Ile Asn Glu Ala Arg Asn Ala Asn Gln Asn Thr Leu IleGlu 1605 1610 1615 Lys Thr Ala Asn Lys Ser Glu Gln Ile Asp Asn Pro ArgThr Glu Ile 1620 1625 1630 Asn Leu Ile Asn Glu Ala Arg Asn Ala Ile AspLys Asn Glu Tyr Ser 1635 1640 1645 Ile Lys Ser Glu Gln Ile Asp Asn ProArg Thr Glu Ile Asn Leu Ile 1650 1655 1660 Asn Glu Ala Arg Asn Ala SerIle Thr Asp Leu Gly Thr Lys Ser Glu 1665 1670 1675 1680 Gln Ile Asp AsnPro Arg Thr Glu Ile Asn Leu Ile Asn Glu Ala Arg 1685 1690 1695 Asn AlaAsn Gln Asn Thr Leu Ile Glu Lys Ser Glu Gln Ile Asp Asn 1700 1705 1710Pro Arg Thr Glu Ile Asn Leu Ile Asn Glu Ala Arg Asn Ala Ala Leu 17151720 1725 His Glu Gln Gln Leu Glu Thr Leu Thr Lys Ser Glu Gln Ile AspAsn 1730 1735 1740 Pro Arg Thr Glu Ile Asn Leu Ile Asn Glu Ala Arg AsnAla Asn Ser 1745 1750 1755 1760 Ser Asp Ser Glu Gln Ile Asp Asn Pro ArgThr Glu Ile Asn Leu Ile 1765 1770 1775 Asn Glu Ala Arg Asn Ala Asn LysAla Asp Ala Asp Ala Ser Phe Glu 1780 1785 1790 Thr Leu Thr Lys Ser GluGln Ile Asp Asn Pro Arg Thr Glu Ile Asn 1795 1800 1805 Leu Ile Asn GluAla Arg Asn Ala Phe Ala Ala Thr Ala Ile Ala Lys 1810 1815 1820 Asp LysSer Glu Gln Ile Asp Asn Pro Arg Thr Glu Ile Asn Leu Ile 1825 1830 18351840 Asn Glu Ala Arg Asn Ala Lys Ala Ser Ser Glu Asn Thr Gln Asn Ile1845 1850 1855 Ala Lys Ser Glu Gln Ile Asp Asn Pro Arg Thr Glu Ile AsnLeu Ile 1860 1865 1870 Asn Glu Ala Arg Asn Ala Arg Leu Leu Asp Gln LysSer Glu Gln Ile 1875 1880 1885 Asp Asn Pro Arg Thr Glu Ile Asn Leu IleAsn Glu Ala Arg Asn Ala 1890 1895 1900 Ala Ala Thr Ala Asp Ala Ile ThrLys Asn Gly Xaa Ser Glu Gln Ile 1905 1910 1915 1920 Asp Asn Pro Arg ThrGlu Ile Asn Leu Ile Asn Glu Ala Arg Asn Ala 1925 1930 1935 Ala Lys AlaXaa Ala Ala Asn Xaa Asp Arg Ser Glu Gln Ile Asp Asn 1940 1945 1950 ProArg Thr Glu Ile Asn Leu Ile Asn Glu Ala Arg Asn Ala Xaa Ala 1955 19601965 Asn Tyr Ala Thr Pro Ser Ile Thr Ile Asn Ser Asn Gln Ala Asp Ile1970 1975 1980 Ala Gln Asn Gln Thr Asp Ile Gln Asp Leu Ala Ala Tyr AsnGlu Leu 1985 1990 1995 2000 Gln Ser Glu Gln Ile Asp Asn Pro Arg Thr GluIle Asn Leu Ile Asn 2005 2010 2015 Glu Ala Arg Asn Ala Asn Gln Ala AspIle Ala Asn Asn Ile Asn Asn 2020 2025 2030 Ile Tyr Glu Leu Ala Gln GlnGln Asp Gln Ser Glu Gln Ile Asp Asn 2035 2040 2045 Pro Arg Thr Glu IleAsn Leu Ile Asn Glu Ala Arg Asn Ala Tyr Asn 2050 2055 2060 Glu Arg GlnThr Glu Ala Ile Asp Ala Leu Asn Ser Glu Gln Ile Asp 2065 2070 2075 2080Asn Pro Arg Thr Glu Ile Asn Leu Ile Asn Glu Ala Arg Asn Ala Ile 20852090 2095 Leu Gly Asp Thr Ala Ile Val Ser Asn Ser Gln Asp Ser Glu GlnIle 2100 2105 2110 Asp Asn Pro Arg Thr Glu Ile Asn Leu Ile Asn Glu AlaArg Asn Ala 2115 2120 2125 Lys Ala Leu Glu Ser Asn Val Glu Glu Gly LeuLeu Asp Leu Ser Gly 2130 2135 2140 Arg Ser Glu Gln Ile Asp Asn Pro ArgThr Glu Ile Asn Leu Ile Asn 2145 2150 2155 2160 Glu Ala Arg Asn Ala AlaLeu Glu Ser Asn Val Glu Glu Gly Leu Leu 2165 2170 2175 Glu Leu Ser GlyArg Thr Ile Asp Gln Arg Ser Glu Gln Ile Asp Asn 2180 2185 2190 Pro ArgThr Glu Ile Asn Leu Ile Asn Glu Ala Arg Asn Ala Asn Gln 2195 2200 2205Ala His Ile Ala Asn Asn Ile Asn Xaa Ile Tyr Glu Leu Ala Gln Gln 22102215 2220 Gln Asp Gln Lys Ser Glu Gln Ile Asp Asn Pro Arg Thr Glu IleAsn 2225 2230 2235 2240 Leu Ile Asn Glu Ala Arg Asn Ala Xaa Ala Asn TyrAla Thr Pro Ser 2245 2250 2255 Ile Thr Ile Asn Asn Gln Ala Asp Ile AlaGln Asn Gln Thr Asp Ile 2260 2265 2270 Gln Asp Leu Ala Ala Tyr Asn GluLeu Gln Ser Glu Gln Ile Asp Asn 2275 2280 2285 Pro Arg Thr Glu Ile AsnLeu Ile Asn Glu Ala Arg Asn Ala Ala Thr 2290 2295 2300 His Asp Tyr AsnGlu Arg Gln Thr Glu Ala Ser Glu Gln Ile Asp Asn 2305 2310 2315 2320 ProArg Thr Glu Ile Asn Leu Ile Asn Glu Ala Arg Asn Ala Lys Ala 2325 23302335 Ser Ser Glu Asn Thr Gln Asn Ile Ala Lys Ser Glu Gln Ile Asp Asn2340 2345 2350 Pro Arg Thr Glu Ile Asn Leu Ile Asn Glu Ala Arg Asn AlaMet Ile 2355 2360 2365 Leu Gly Asp Thr Ala Ile Val Ser Asn Ser Gln AspAsn Lys Thr Gln 2370 2375 2380 Leu Lys Phe Tyr Lys Ser Glu Gln Ile AspAsn Pro Arg Thr Glu Ile 2385 2390 2395 2400 Asn Leu Ile Asn Glu Ala ArgAsn Ala Ala Gly Asp Thr Ile Ile Pro 2405 2410 2415 Leu Asp Asp Asp XaaXaa Pro Ser Glu Gln Ile Asp Asn Pro Arg Thr 2420 2425 2430 Glu Ile AsnLeu Ile Asn Glu Ala Arg Asn Ala Xaa Ala Asn Tyr Ala 2435 2440 2445 ThrPro Ser Ile Thr Ile Asn Ser Leu Leu His Glu Gln Gln Leu Xaa 2450 24552460 Gly Lys Ser Glu Gln Ile Asp Asn Pro Arg Thr Glu Ile Asn Leu Ile2465 2470 2475 2480 Asn Glu Ala Arg Asn Ala Xaa Ala Asn Tyr Ala Thr ProSer Ile Thr 2485 2490 2495 Ile Asn Ile Phe Phe Asn Xaa Gly Ser Glu GlnIle Asp Asn Pro Arg 2500 2505 2510 Thr Glu Ile Asn Leu Ile Asn Glu AlaArg Asn Ala Xaa Ala Asn Tyr 2515 2520 2525 Ala Thr Pro Ser Ile Thr IleAsn Asn Asn Ile Asn Asn Ile Tyr Glu 2530 2535 2540 Leu Ala Gln Gln GlnAsp Gln His Ser Ser Asp Ile Lys Thr Leu Ser 2545 2550 2555 2560 Glu GlnIle Asp Asn Pro Arg Thr Glu Ile Asn Leu Ile Asn Glu Ala 2565 2570 2575Arg Asn Ala Gly Gly Thr Gly Cys Ala Gly Gly Thr Cys Ala Gly Ala 25802585 2590 Thr Cys Ala Gly Thr Gly Ala Cys Ser Glu Gln Ile Asp Asn AspAsn 2595 2600 2605 Ala Leu Ile Asn Glu Ala Arg Asp Asx Leu Glu Gly CysCys Ala Cys 2610 2615 2620 Cys Ala Ala Cys Cys Ala Ala Gly Cys Thr GlyAla Cys Ser Glu Gln 2625 2630 2635 2640 Ile Asp Asn Asp Asn Ala Leu IleAsn Glu Ala Arg Asp Asx Leu Glu 2645 2650 2655 Ala Gly Cys Gly Gly ThrCys Gly Cys Cys Thr Gly Cys Thr Thr Gly 2660 2665 2670 Ala Thr Cys AlaGly Ser Glu Gln Ile Asp Asn Asp Asn Ala Leu Ile 2675 2680 2685 Asn GluAla Arg Asp Asx Leu Glu Cys Thr Gly Ala Thr Cys Ala Ala 2690 2695 2700Gly Cys Ala Gly Gly Cys Gly Ala Cys Cys Gly Cys Thr Ser Glu Gln 27052710 2715 2720 Ile Asp Asn Asp Asn Ala Leu Ile Asn Glu Ala Arg Asp AsxLeu Glu 2725 2730 2735 Cys Ala Ala Gly Ala Thr Cys Thr Gly Gly Cys CysGly Cys Thr Thr 2740 2745 2750 Ala Cys Ala Ala Ser Glu Gln Ile Asp AsnAsp Asn Ala Leu Ile Asn 2755 2760 2765 Glu Ala Arg Asp Asx Leu Glu ThrThr Gly Thr Ala Ala Gly Cys Gly 2770 2775 2780 Gly Cys Cys Ala Gly AlaThr Cys Thr Thr Gly Ser Glu Gln Ile Asp 2785 2790 2795 2800 Asn Asp AsnAla Leu Ile Asn Glu Ala Arg Asp Asx Leu Glu Thr Gly 2805 2810 2815 CysAla Thr Gly Ala Gly Cys Cys Gly Cys Ala Ala Ala Cys Cys Cys 2820 28252830 Ser Glu Gln Ile Asp Asn Asp Asn Ala Leu Ile Asn Glu Ala Arg Asp2835 2840 2845 Asx Leu Glu Leu Leu Ala Glu Gln Gln Leu Asn Gly Ser GluGln Ile 2850 2855 2860 Asp Asn Pro Arg Thr Glu Ile Asn Leu Ile Asn GluAla Arg Asn Ala 2865 2870 2875 2880 Ala Leu Glu Ser Asn Val Glu Glu GlyLeu Ser Glu Gln Ile Asp Asn 2885 2890 2895 Pro Arg Thr Glu Ile Asn LeuIle Asn Glu Ala Arg Asn Ala Ala Leu 2900 2905 2910 Glu Ser Asn Val GluGlu Gly Leu Leu Asp Leu Ser Ser Glu Gln Ile 2915 2920 2925 Asp Asn ProArg Thr Glu Ile Asn Leu Ile Asn Glu Ala Arg Asn Ala 2930 2935 2940 AsnAla Lys Ala Ser Ala Ala Asn Thr Asp Arg Ser Glu Gln Ile Asp 2945 29502955 2960 Asn Pro Arg Thr Glu Ile Asn Leu Ile Asn Glu Ala Arg Asn AlaAla 2965 2970 2975 Ala Thr Ala Ala Asp Ala Ile Thr Lys Asn Gly Asn SerGlu Gln Ile 2980 2985 2990 Asp Asn Pro Arg Thr Glu Ile Asn Leu Ile AsnGlu Ala Arg Asn Ala 2995 3000 3005 Ser Ile Thr Asp Leu Gly Thr Lys ValAsp Gly Phe Asp Gly Arg Ser 3010 3015 3020 Glu Gln Ile Asp Asn Pro ArgThr Glu Ile Asn Leu Ile Asn Glu Ala 3025 3030 3035 3040 Arg Asn Ala ValAsp Ala Leu Xaa Thr Lys Val Asn Ala Leu Asp Xaa 3045 3050 3055 Lys ValAsn Ser Glu Gln Ile Asp Asn Pro Arg Thr Glu Ile Asn Leu 3060 3065 3070Ile Asn Glu Ala Arg Asn Ala Xaa Ala Asn Tyr Ala Thr Pro Ser Ile 30753080 3085 Thr Ile Asn Ser Ala Ala Gln Ala Ala Leu Ser Gly Leu Phe ValPro 3090 3095 3100 Tyr Ser Val Gly Lys Phe Asn Ala Thr Ala Ala Leu GlyGly Tyr Gly 3105 3110 3115 3120 Ser Lys Ser Glu Gln Ile Asp Asn Pro ArgThr Glu Ile Asn Leu Ile 3125 3130 3135 Asn Glu Ala Arg Asn Ala Ser GlyArg Leu Leu Asp Gln Lys Ala Asp 3140 3145 3150 Ser Glu Gln Ile Asp AsnPro Arg Thr Glu Ile Asn Leu Ile Asn Glu 3155 3160 3165 Ala Arg Asn AlaGln Lys Ala Asp Ile Asp Asn Asn Ile Asn Ser Glu 3170 3175 3180 Gln IleAsp Asn Pro Arg Thr Glu Ile Asn Leu Ile Asn Glu Ala Arg 3185 3190 31953200 Asn Ala Asn Asn Ile Asn Asn Ile Tyr Glu Leu Ala Ser Glu Gln Ile3205 3210 3215 Asp Asn Pro Arg Thr Glu Ile Asn Leu Ile Asn Glu Ala ArgAsn Ala 3220 3225 3230 Asn Asn Ile Tyr Glu Leu Ala Gln Gln Gln Ser GluGln Ile Asp Asn 3235 3240 3245 Pro Arg Thr Glu Ile Asn Leu Ile Asn GluAla Arg Asn Ala Ala Gln 3250 3255 3260 Gln Gln Asp Gln His Ser Ser AspSer Glu Gln Ile Asp Asn Pro Arg 3265 3270 3275 3280 Thr Glu Ile Asn LeuIle Asn Glu Ala Arg Asn Ala Gln Asp Gln His 3285 3290 3295 Ser Ser AspIle Lys Thr Ser Glu Gln Ile Asp Asn Pro Arg Thr Glu 3300 3305 3310 IleAsn Leu Ile Asn Glu Ala Arg Asn Ala His Ser Ser Asp Ile Lys 3315 33203325 Thr Leu Lys Asn Ser Glu Gln Ile Asp Asn Pro Arg Thr Glu Ile Asn3330 3335 3340 Leu Ile Asn Glu Ala Arg Asn Ala Asp Ile Lys Thr Leu LysAsn Asn 3345 3350 3355 3360 Val Glu Ser Glu Gln Ile Asp Asn Pro Arg ThrGlu Ile Asn Leu Ile 3365 3370 3375 Asn Glu Ala Arg Asn Ala Thr Leu LysAsn Asn Val Glu Glu Gly Leu 3380 3385 3390 Ser Glu Gln Ile Asp Asn ProArg Thr Glu Ile Asn Leu Ile Asn Glu 3395 3400 3405 Ala Arg Asn Ala GluGlu Gly Leu Leu Asp Leu Ser Gly Arg Ser Glu 3410 3415 3420 Gln Ile AspAsn Pro Arg Thr Glu Ile Asn Leu Ile Asn Glu Ala Arg 3425 3430 3435 3440Asn Ala Leu Ser Gly Arg Leu Ile Asp Gln Lys Ala Ser Glu Gln Ile 34453450 3455 Asp Asn Pro Arg Thr Glu Ile Asn Leu Ile Asn Glu Ala Arg AsnAla 3460 3465 3470 Asp Gln Lys Ala Asp Ile Ala Lys Asn Gln Ser Glu GlnIle Asp Asn 3475 3480 3485 Pro Arg Thr Glu Ile Asn Leu Ile Asn Glu AlaArg Asn Ala Ala Lys 3490 3495 3500 Asn Gln Ala Asp Ile Ala Gln Asn SerGlu Gln Ile Asp Asn Pro Arg 3505 3510 3515 3520 Thr Glu Ile Asn Leu IleAsn Glu Ala Arg Asn Ala Ile Ala Gln Asn 3525 3530 3535 Gln Thr Asp IleGln Asp Ser Glu Gln Ile Asp Asn Pro Arg Thr Glu 3540 3545 3550 Ile AsnLeu Ile Asn Glu Ala Arg Asn Ala Asp Ile Gln Asp Leu Ala 3555 3560 3565Ala Tyr Asn Glu Ser Glu Gln Ile Asp Asn Pro Arg Thr Glu Ile Asn 35703575 3580 Leu Ile Asn Glu Ala Arg Asn Ala Cys Gly Gly Gly Ala Thr CysCys 3585 3590 3595 3600 Gly Thr Gly Ala Ala Gly Ala Ala Ala Ala Ala ThrGly Cys Cys Gly 3605 3610 3615 Cys Ala Gly Gly Thr Ser Glu Gln Ile AspAsn Asp Asn Ala Leu Ile 3620 3625 3630 Asn Glu Ala Arg Asp Asx Leu GluCys Gly Gly Gly Ala Thr Cys Cys 3635 3640 3645 Cys Gly Thr Cys Gly CysAla Ala Gly Cys Cys Gly Ala Thr Thr Gly 3650 3655 3660 Ser Glu Gln IleAsp Asn Asp Asn Ala Leu Ile Asn Glu Ala Arg Asp 3665 3670 3675 3680 AsxLeu Glu Ser Gly Arg Leu Leu Asp Gln Lys Ala Asp Ile Asp Asn 3685 36903695 Asn Ile Asn Asn Ile Tyr Glu Leu Ala Gln Gln Gln Asp Gln His Ser3700 3705 3710 Ser Asp Ile Lys Thr Leu Lys Asn Asn Val Glu Glu Gly LeuLeu Asp 3715 3720 3725 Leu Ser Gly Arg Leu Ile Asp Gln Lys Ala Asp IleAla Lys Asn Gln 3730 3735 3740 Ala Asp Ile Ala Gln Asn Gln Thr Asp IleGln Asp Leu Ala Ala Tyr 3745 3750 3755 3760 Asn Glu Ser Glu Gln Ile AspAsn Pro Arg Thr Glu Ile Asn Leu Ile 3765 3770 3775 Asn Glu Ala Arg AsnAla Ala Trp Glx Xaa Asp Cys 3780 3785 77 13 PRT Moraxella catarrhalis 77Ala Ala Thr Ala Ala Asp Ala Ile Thr Lys Asn Gly Asn 1 5 10 78 15 PRTMoraxella catarrhalis 78 Ser Ile Thr Asp Leu Gly Thr Lys Val Asp Gly PheAsp Gly Arg 1 5 10 15 79 16 PRT Moraxella catarrhalis MOD_RES (5)..(13)Xaa = any 79 Val Asp Ala Leu Xaa Thr Lys Val Asn Ala Leu Asp Xaa Lys ValAsn 1 5 10 15 80 30 PRT Moraxella catarrhalis 80 Ala Ala Gln Ala Ala LeuSer Gly Leu Phe Val Pro Tyr Ser Val Gly 1 5 10 15 Lys Phe Asn Ala ThrAla Ala Leu Gly Gly Tyr Gly Ser Lys 20 25 30 81 10 PRT Moraxellacatarrhalis 81 Ser Gly Arg Leu Leu Asp Gln Lys Ala Asp 1 5 10 82 10 PRTMoraxella catarrhalis 82 Gln Lys Ala Asp Ile Asp Asn Asn Ile Asn 1 5 1083 10 PRT Moraxella catarrhalis 83 Asn Asn Ile Asn Asn Ile Tyr Glu LeuAla 1 5 10 84 10 PRT Moraxella catarrhalis 84 Asn Asn Ile Tyr Glu LeuAla Gln Gln Gln 1 5 10 85 10 PRT Moraxella catarrhalis 85 Ala Gln GlnGln Asp Gln His Ser Ser Asp 1 5 10 86 10 PRT Moraxella catarrhalis 86Gln Asp Gln His Ser Ser Asp Ile Lys Thr 1 5 10 87 10 PRT Moraxellacatarrhalis 87 His Ser Ser Asp Ile Lys Thr Leu Lys Asn 1 5 10 88 10 PRTMoraxella catarrhalis 88 Asp Ile Lys Thr Leu Lys Asn Asn Val Glu 1 5 1089 10 PRT Moraxella catarrhalis 89 Thr Leu Lys Asn Asn Val Glu Glu GlyLeu 1 5 10 90 10 PRT Moraxella catarrhalis 90 Glu Glu Gly Leu Leu AspLeu Ser Gly Arg 1 5 10 91 10 PRT Moraxella catarrhalis 91 Leu Ser GlyArg Leu Ile Asp Gln Lys Ala 1 5 10 92 10 PRT Moraxella catarrhalis 92Asp Gln Lys Ala Asp Ile Ala Lys Asn Gln 1 5 10 93 10 PRT Moraxellacatarrhalis 93 Ala Lys Asn Gln Ala Asp Ile Ala Gln Asn 1 5 10 94 10 PRTMoraxella catarrhalis 94 Ile Ala Gln Asn Gln Thr Asp Ile Gln Asp 1 5 1095 10 PRT Moraxella catarrhalis 95 Asp Ile Gln Asp Leu Ala Ala Tyr AsnGlu 1 5 10 96 29 DNA Moraxella catarrhalis 96 cgggatccgt gaagaaaaatgccgcaggt 29 97 24 DNA Moraxella catarrhalis 97 cgggatcccg tcgcaagccgattg 24 98 79 PRT Moraxella catarrhalis 98 Ser Gly Arg Leu Leu Asp GlnLys Ala Asp Ile Asp Asn Asn Ile Asn 1 5 10 15 Asn Ile Tyr Glu Leu AlaGln Gln Gln Asp Gln His Ser Ser Asp Ile 20 25 30 Lys Thr Leu Lys Asn AsnVal Glu Glu Gly Leu Leu Asp Leu Ser Gly 35 40 45 Arg Leu Ile Asp Gln LysAla Asp Ile Ala Lys Asn Gln Ala Asp Ile 50 55 60 Ala Gln Asn Gln Thr AspIle Gln Asp Leu Ala Ala Tyr Asn Glu 65 70 75

What is claimed is:
 1. An isolated nucleic acid encoding the UspA2antigen (SEQ ID NO:3) of the M. catarrhalis isolate O35E.
 2. An isolatednucleic acid having the uspA2 DNA sequence (SEQ ID NO:4) of the M.catarrhalis isolate O35E.